This work was supported by National Institutes of Health grants K01HL130497, R01HL147818, R01HL144941, and R03HL155041 to A.K. We thank the Digital Histology Shared Resource - Vanderbilt Health Nashville, TN https://www.vumc.org/dhsr/46298&#160;for visualization and slide scanning.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee approved all procedures described in this manuscript. Mice are housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals. All subjects gave written informed consent before enrolling in the study as approved by the Institutional Review Board of Vanderbilt University. All procedures were performed according to the Declaration of Helsinki.
1. Preparation of plasmids encoding D11-alkaline phosphatase fusion protein and negative control vectors
<ol>
	<li>Construct a modified version of the pCANTAB5E plasmid13,14 in which the single chain fragment variable (scFv) segment is linked at its 3&#39; end to a sequence encoding bacterial alkaline phosphatase (AP). 
	NOTE: Immediately downstream of the scFv sequence&#39;s NotI restriction site, the modified plasmid no longer contains the coding sequence of the E-tag and instead encodes the linker sequence GGSGGHMGSGG, followed by the sequence for AP (GenBank accession number AXY87039.1, T16-K464)15,16. Downstream of AP, the plasmid will encode 8xHis and DYKDDDDK tags. At the 3&#39; end of the tags, the coding sequence ends with an Amber stop codon. The modified plasmid is called pCANTAB5-AP.</li>
	<li>Clone D11 scFv (GenBank accession number AAW28931.1) into pCANTAB5-AP with the SfiI/NotI restriction sites.</li>
	<li>Clone D20 scFv into pCANTAB5-AP with the SfiI/NotI restriction sites. 
	NOTE: D20 scFv is a negative control designed to assess the tissue interaction pattern of an irrelevant scFv. This scFv was originally selected by phage display for its ability to interact with a glycan group known as A2.</li>
	<li>Generate an &#34;empty&#34; vector by removing the scFv portion from the plasmid entirely and replacing it with an &#34;AP linker&#34; coding sequence consisting of amino acids GGGGSGRAGSGGGGS.</li>
	<li>Transform all plasmids into competent TG1 E. coli and grow bacteria overnight at 30 &#176;C, on 0.5% agar plates containing 2xYT supplemented with 100 &#181;g/mL Ampicillin and 2% glucose (2xYTAG) medium. 
	NOTE: Strains with the supE gene (such as TG1 E. coli) do not suppress the amber stop codon 100%. Estimates range from 0.8 - 20% of the time, so there is still a lot of product that terminates just downstream of BAP, as it is intended for these experiments17. TG1 E. coli are mainly used for practical reasons, such as the reduction of time and costs, their compatibility with the pCANTAB protein expression system, and their use in phage display, which is commonly conducted in the lab. Downstream of the amber stop codon following BAP is a sequence for geneIII. GeneIII fusion proteins need to be expressed rarely for phage display to function as intended because scFv-geneIII fusion proteins interfere with that particular geneIII protein to re-infect na&#239;ve bacteria. Therefore, fusion-free geneIII proteins need to exist during virion assembly for the generation of functioning phage i.e., scFv-geneIII protein products are usually relatively rare inside the bacterium. D11-AP, D20-AP, empty vector, and all constructs used in this article are all affected similarly by the occasional geneIII fusion protein expression. There are still observed differences in how these proteins behave.</li>
	<li>Pick an individual colony and inoculate a fresh 5 mL of 2xYTAG culture. Allow bacteria to grow overnight at 30 &#176;C and shaking at 150 rpm.</li>
	<li>Pellet the cells by centrifugation at 3,000 x g for 10 min at room temperature. Discard the supernatant and resuspend the pellet in 2 mL of 85% 2xYTAG and 15% glycerol.</li>
	<li>Maintain glycerol stocks in a -80 &#176;C freezer.</li>
</ol>
2. Protein expression and generation of periplasmic extract
NOTE: Generation of the periplasmic extract is a commonly used method for protein expression in phage display, mainly because the formation of disulfide bonds is important in scFv and antibody generation. The method avoids the need to generate lysates (commonly containing inclusion bodies) and ensures that proteins are properly folded. pCANTAB has a gIII signal sequence upstream of the scFv portion of the D11-BAP fusion protein. The signal sequence ensures that the protein is shuttled to the periplasmic space of the bacterium, and then the signal sequence is cleaved. The periplasmic space provides an oxidizing environment, which is crucial to the proper formation of disulfide bridges. Osmotic shock is used to derive periplasmic extracts because it disrupts the outer membrane enough to release the periplasmic proteins into the surrounding medium, while keeping the bacterium intact.
<ol>
	<li>Inoculate TG1 E. coli glycerol stocks containing the relevant plasmids into a 60 mL culture of 2xYTAG and culture overnight at 30 &#176;C with shaking at 150 rpm.</li>
	<li>To induce protein expression, pellet bacterial cultures at 3,000 x g for 10 min, resuspend in 60 mL of 2xYTA medium, and culture overnight at 30 &#176;C with shaking at 150 rpm. 
	NOTE: The switch from 2xTYAG to 2xYTA medium helps in sufficient induction of protein expression18. When glucose is present in the bacterial medium, the lac promoter is suppressed because bacteria preferentially consume glucose and ignore lactose, as glucose takes less energy to process. When glucose is removed following the medium switch, bacteria rely on the carbohydrates provided by the 2xYT medium. Yeast extract (the Y component in 2xYT) contains carbohydrates, among them lactose, and is, therefore, able to drive protein expression through the lac promoter. IPTG is not necessary with the pCANTAB construct and can result in excessive protein expression, along with inclusion bodies that result in non-functional protein.</li>
	<li>Generate periplasmic extracts via osmotic shock.
	<ol>
		<li>Pellet bacterial cultures at 3,000 x g for 10 min.</li>
		<li>Resuspend in 20 mL of 1xTES (0.2 M Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5 M sucrose) and incubate for 1 h on ice.</li>
		<li>Pellet again at 3,000 x g for 10 min, and resuspend in 15 mL of 0.05 M Tris, pH 7.6.</li>
	</ol>
	</li>
	<li>Incubate this suspension on ice for 1 h, then clarify by centrifugation at 5,000 x g for 10 min.</li>
	<li>Transfer supernatants to a fresh tube and store at -20 &#176;C until needed for either purification or direct use in experiments.</li>
	<li>Assess the cell lysis by the presence of active D11-BAP in the ELISA. Add 3-5 &#181;L of the periplasmic extract to one mL of pNPP, then observe if a color change happens over the next 10 min (color goes from clear-yellow to intense yellow). 
	&#8203;NOTE: The color can be compared side-by-side to a tube of pNPP which has not received any lysate or a tube of pNPP which received lysate of na&#239;ve TG1 bacteria. Quantify with absorbance at 405 nm.</li>
</ol>
4. Characterization of D11-AP titer in ELISA
<ol>
	<li>Coat a 384-well polystyrene plate overnight at 4 &#176;C with 25 &#181;L/well phosphate-buffered saline (PBS) (1.8 mM KH2PO4, 10 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl) containing either 5 &#181;g/mL of mouse serum albumin (MSA) (negative control), or 5 &#181;g/mL of IsoLG/MSA (positive control).</li>
	<li>Empty the plate and tap dry. Wash once with PBS + 0.1% Tween (PBS-T). Empty the plate and tap dry.</li>
	<li>Fill the plate with PBS-T as a blocking buffer (120 &#181;L/well). Incubate for 1 h at room temperature.</li>
	<li>Empty the plate and tap dry. Apply serial 1:2 dilutions of D11-AP periplasmic extracts at 25 &#181;L/well and diluted in PBS-T. Include one well containing only PBS-T as a negative control. 
	NOTE: Typical dilution ranges for periplasmic extracts are 1:8 - 1:4096. For a 25 &#181;L assay volume, start with 50 &#181;L of the starting &#34;1:8&#34; concentration: 6.25 &#181;L periplasmic extract and 43.75 &#181;L PBS-T. Then, perform a 2-fold dilution by removing 25 &#181;L of this solution and adding it to the next well containing 25 &#181;L of PBS-T and pipette it up and down. This well now contains the &#34;1:16&#34; dilution. Keep repeating the 2-fold dilution to generate the described 1:2 dilution series.</li>
	<li>Incubate for 1.5 h at room temperature.</li>
	<li>Empty the plate and tap dry. Wash 5 times with PBS-T. Empty and tap dry.</li>
	<li>Prepare pNPP solution by dissolving 1 g of pNPP in 1 L of 930 mM diethanolamine (98% stock solution diluted 1:10 in H2O), with 0.5 mM MgCl2 and adjusted to pH 9.5 with HCl.</li>
	<li>Apply 25 &#181;L/well pNPP solution to develop AP. Incubate for 1 h at room temperature and determine the absorbance at 405 nm within each well using a compatible plate reader.</li>
	<li>Compare the signal generated from the IsoLG/MSA wells to the noise generated from the MSA wells and find the range of dilutions in which the signal is at least 5-fold higher than the noise.</li>
	<li>Plot this D11-AP signal dilution series on a graph, determine the linear range of the curve, and establish the dilution where 50% of the signal can be observed. 
	&#8203;NOTE: If this dilution is equivalent to approximately 1:1,000, then the D11-AP solution may be used at a concentration of 1:10 in IHC/IF.</li>
</ol>
5. Immunofluorescence
<ol>
	<li>Cut serial sections of mouse and human paraffin-embedded tissues (5 &#181;m thick) using a microtome and place in a warm water bath (37 &#176;C). Mount tissue sections on glass slides and allow to dry overnight. 
	NOTE: For this study, aortae were obtained from mice. Colon sections of normotensive and hypertensive humans were obtained from the Vanderbilt Cooperative Human Tissue Network.</li>
	<li>Immerse slides in xylene three times for 5 min to deparaffinize tissues.</li>
	<li>Rehydrate tissues in 2 washes each of 95%, 70% and 50% ethanol in H2O.</li>
	<li>Wash slides in Tris-buffered saline (TBS) with 0.1% Tween20 (TBST) three times with quick washes by filling the slide holder with TBST then discarding TBST. 
	NOTE: Hydrated slides can be stored in TBST at 4 &#176;C for no longer than a week before antigen retrieval.</li>
	<li>To perform heat-induced antigen retrieval of slides, place slides in pre-heated (80-95 &#176;C) sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) and incubate in a pressure cooker set to 4 min on high pressure for a total antigen retrieval time of 20 min.</li>
	<li>Remove slides from the pressure cooker and allow them to cool for 20 min to room temperature.</li>
	<li>Wash slides in TBST three times with quick washes. 
	NOTE: Slides can be stored in TBST after antigen retrieval for no longer than a week before staining.</li>
	<li>Add 2% BSA dissolved in TBST to block slides. Cover slides with a strip of paraffin film and incubate at room temperature for 15 min.</li>
	<li>Discard the blocking buffer from the slides.</li>
	<li>Add 200 &#181;L of 1:10 D11-AP in TBST to the slides and cover with a strip of paraffin film.</li>
	<li>Incubate in a humidified chamber to minimize the antibody solution evaporation for 3 h at room temperature.</li>
	<li>Wash slides three times in TBST.</li>
	<li>Develop with a colorimetric or fluorescent alkaline phosphatase developer for immunohistochemistry or immunofluorescence, respectively. Wash slides once with TBST to remove excess developer and prevent further color development.</li>
	<li>Counterstain slides with Hoechst nuclear stain at 1 &#181;g/mL in PBS for immunofluorescence. Wash slides once in TBST to remove any excess counterstain.</li>
	<li>Apply coverslips using the mounting medium.</li>
	<li>View slides under an inverted light microscope for immunohistochemistry or a confocal fluorescent microscope for immunofluorescence.</li>
</ol>
6. Negative controls
NOTE: Four negative control experiments can be performed to confirm the specificity of D11-AP staining for IsoLG. Negative control experiments should be performed in the same staining set under the same conditions.
<ol>
	<li>In the first negative control experiment, incubate tissues with D11-AP diluted in TBST or TBST alone.</li>
	<li>Incubate tissues with diluted D11-AP in TBST and bacterial periplasmic extract without D11-AP (AP Linker) diluted in TBST.</li>
	<li>Perform a competitive assay with IsoLG/MSA or non-adducted MSA as previously described12.
	<ol>
		<li>Prepare IsoLG and IsoLG adducted to mouse serum albumin (MSA) as previously described19, at a molar ratio of 8 IsoLG: 1 MSA (8:1 IsoLG/MSA).</li>
		<li>Dilute D11-AP 1:10 in TBST.</li>
		<li>Incubate diluted D11-AP with 50 &#181;g/mL IsoLG/MSA or non-adducted MSA for 1 h at room temperature.</li>
		<li>Add D11-AP with IsoLG/MSA or D11-AP with non-adducted MSA to tissues for staining.</li>
	</ol>
	</li>
	<li>Use irrelevant scFv antibody, D20, to stain tissues for the final negative control set.</li>
</ol>

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The authors have nothing to disclose.

D11 has been used extensively to detect IsoLG-adducted proteins in cells or tissues as a marker for inflammation or oxidative stress in disease8,9,20. Previously, D11 contained an E tag and IHC development required the use of a secondary anti-E tag antibody conjugated with HRP10,20,21. Here, we have developed and optimized protocol for detection of IsoLG-adducted proteins using the D11 antibody conjugated with alkaline phosphatase in place of the E-tag, which eliminates the need for a secondary antibody incubation.
To determine the specificity of D11-AP, four negative control experiments were performed. We performed the protocol without the presence of D11 and had minimal development. These results have a two-fold indication: endogenous alkaline phosphatase is not contributing to development, and the staining observed is due to D11 and not another contributing factor. Next, we stained slides with the AP linker without D11. This experiment resulted in little staining, which indicates free AP or other factors in the periplasmic extract is not causing the stain we observe in the presence of D11. To ensure the specificity of D11 to IsoLG, we preincubated D11-AP with purified IsoLG before staining slides. We saw a decrease in development which indicates that the D11-AP was bound to IsoLG protein, thus exhausting the amount of free D11-AP to bind to IsoLG present in the tissue. Finally, to ensure D11-AP was binding to IsoLG and not the MSA protein IsoLG was bound to, we preincubated D11-AP with MSA only. There were no changes in development, indicating D11-AP was not binding to MSA but the IsoLG protein. Lastly, researchers developing the staining protocol were blind to hypertensive status of human intestinal tissue. The differences in staining observed between patients with hypertension and patients with normotension were not due to bias and have been previously described22,23.
Although our protocol for detection of IsoLG-adducted proteins using the D11 antibody conjugated with alkaline phosphatase in place of the E-tag is rigorous and robust and eliminates the need for a secondary antibody incubation, it has some limitations. One limitation is that we used D11 conjugated with alkaline phosphatase in the periplasmic extract, and there could be false staining of endogenous alkaline phosphatase in the periplasmic extract or certain tissues, such as intestine24. However, the first step to develop this protocol included disabling endogenous alkaline phosphatase that may be present in tissues25. Initially, cold acetic acid, BME, and Levamisole26 were tested for efficiency. None of these completely diminished the presence of active endogenous alkaline phosphatase. Heat has been used to deactivate alkaline phosphatase27, so we tested heat deactivation of alkaline phosphatase in different buffers. We found heating mounted and hydrated slides in citrate buffer eliminated most endogenous alkaline phosphatase. Slides were developed initially using a Chemiluminescent/Fluorescent Substrate, but when imaged without this substrate, there was a high amount of autofluorescence. VectorRed is a substrate which develops in the presence of alkaline phosphatase to produce a chromogen which can be visualized in the Texas Red/TRITC channel range. Using this substrate, we were able to more easily observe signal above background autofluorescence. Care should be taken during the staining process to minimize artifactual staining. Drying of tissues on slides after hydration until imaging has resulted in heightened development. D11-AP should be aliquoted and stored at -20 &#176;C. Multiple freeze-thaw cycles should be avoided when working with D11-AP. Phosphate-buffered saline (PBS) can also affect the enzymatic activity of alkaline phosphatase and should not be used as a wash buffer28. As with any antibody-based approach, thorough testing and optimization must be carried out to ensure that staining is specific, and that signal is not over or under amplified.
In conclusion, we have developed a powerful, rigorous, and robust optimized protocol to detect IsoLG-adducted proteins using the D11 antibody conjugated with alkaline phosphatase in place of the E-tag. This protocol presents several advantages: First, using D11 as an alkaline phosphatase fusion protein is cheaper. D11 was originally derived from a phage antibody library that could not be commercialized and was expensive to purify. Although D11 in E. coli periplasmic extract could provide an inexpensive alternative, it was ineffective in most assays. Second, the alkaline phosphatase fusion approach allows D11 scfv to have a useful reporter15 (alkaline phosphatase) fused to it and would not need to be purified for use in immunoassays as substrates are commercially available. Third, the E. coli alkaline phosphatase forms dimers29. So D11, when fused to the alkaline phosphatase would also form dimers and this increase the antibody&#39;s avidity and binding activity30. Finally, D11 conjugated with alkaline phosphatase in periplasmic extract can easily be cleaned using Cibacron Blue Sepharose. D11 has a high isoelectric point (~9.2 pH). As such, it is positively charged and can bind to Cibacron Blue through pi-cation interactions. Most of the impurities in the E. coli periplasmic extract can be eluted off the resin. The D11 conjugated with alkaline phosphatase can then be eluted using high salt (~1.5M NaCl) in water. The eluted D11 conjugated with alkaline phosphatase is quite stable at 4-8&#160;&#176;C in the high salt solution. Thus, we have developed a protocol which not only makes the D11 antibody available at a low cost, but also eliminates the extra steps and need for the secondary antibody incubation. This protocol facilitates reproducible measurement of IsoLGs, which accumulate in tissues in multiple diseases where increased oxidative stress plays a role.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62603">Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence</a>. The Authors section was updated from:
Cassandra Warden1 
Alan J. Simmons2 
Lejla Pasic3 
Sean S. Davies4 
Justin H. Layer5 
Raymond L. Mernaugh3 
Annet Kirabo4,6 
1Vanderbilt Eye Institute, Vanderbilt University Medical Center 
2Department of Cell and Developmental Biology, Vanderbilt University 
3Department of Biochemistry, Vanderbilt University 
4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center 
5Division of Hematology and Oncology, Indiana University School of Medicine 
6Department of Molecular Physiology and Biophysics, Vanderbilt University
to:
Cassandra Warden1 
Alan J. Simmons2 
Lejla Pasic3 
Ashley Pitzer4,6 
Sean S. Davies4 
Justin H. Layer5 
Raymond L. Mernaugh3 
Annet Kirabo4,6 
1Vanderbilt Eye Institute, Vanderbilt University Medical Center 
2Department of Cell and Developmental Biology, Vanderbilt University 
3Department of Biochemistry, Vanderbilt University 
4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center 
5Division of Hematology and Oncology, Indiana University School of Medicine 
6Department of Molecular Physiology and Biophysics, Vanderbilt University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62603">Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence</a>. The Authors section was updated.

Erratum: Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs), also known as isoketals, are isomers of the 4-ketoaldehyde family, which are products of lipid peroxidation, and react with and adduct to primary amines on proteins1,2. IsoLGs have been implicated in several diseases, including cardiovascular, Alzheimer&#39;s, lung and liver diseases, and many types of cancer3. IsoLGs have been most extensively studied in their contribution to cardiovascular disease (CVD), which is a significant health and economic burden globally, including the United States. It is estimated that 92.1 million US adults have at least one type of CVD, with 2030 estimated projections reaching to 43.9% of the US adult population4. Lowering blood pressure, cholesterol, and smoking cessation reduces the overall risk and occurrence of CVD events5.
High blood pressure or hypertension is a major risk factor for cardiovascular disease and affects approximately half of the US population6. Previous studies have found that inflammation is an underlying cause of hypertension and that IsoLGs play a role7. Hypertensive stimuli, including angiotensin II, catecholamines, aldosterone, and excess dietary salt, induce IsoLG accumulation in antigen presenting cells including dendritic cells (DCs), which in turn activate T cells to proliferate and produce inflammatory cytokines that contribute to hypertension8,9.
Previously, IsoLGs have been measured by immunohistochemistry, mass spectrometry, enzyme-linked immunosorbent assay, and flow cytometry10,11. To facilitate the measurement of IsoLGs, a single-chain fragment variable (scfv) recombinant antibody (D11) was developed against IsoLGs12. Initially, this D11 antibody contained an 11 amino acid E-tag and required a secondary antibody for immunohistochemistry detection11. However, it was difficult to find a reliable secondary antibody against the E-tag after the discontinuation of its production by the manufacturer. Therefore, we have developed a reliable protocol for immunofluorescent staining of IsoLGs using D11 conjugated with alkaline phosphatase (D11-AP), which we have demonstrated in mouse and human tissues with and without hypertension.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>1 ml TALON HiTrap column (Cobalt-CMA)</td>
			<td>Cytiva</td>
			<td>28953766</td>
			<td></td>
		</tr>
		<tr>
			<td>200 Proof Ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
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			<td>2xYT powder</td>
			<td>MP Biomedicals</td>
			<td>3012-032</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well, clear, flat-bottom polystyrene microplates</td>
			<td>ThermoFisher (NUNC)</td>
			<td>242757</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP)</td>
			<td>Carbosynth</td>
			<td>EN08508</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3indoxyl phosphate, p-toluidine salt (BCIP)</td>
			<td>Carbosynth</td>
			<td>EB09335</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin, sodium salt</td>
			<td>Research Products International (RPI)</td>
			<td>A40040</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically competent TG1 E. coli</td>
			<td>Amid Biosciences</td>
			<td>TG1-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethanolamine, &#62;98%</td>
			<td>Sigma-Aldrich</td>
			<td>D8885</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>Mouting medium</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Research Products International (RPI)</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoclear</td>
			<td>National Diagnostics</td>
			<td>HS-200</td>
			<td>Xylene alternative</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>stock solution = 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl), 30%, Macron Fine Chemicals</td>
			<td>ThermoFisher</td>
			<td>MK-2624-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Research Products International (RPI)</td>
			<td>I52000</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Serum Albumin (MSA)</td>
			<td>Sigma-Aldrich/Calbiochem</td>
			<td>126674</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium chloride (NBT)</td>
			<td>Carbosynth</td>
			<td>EN13587</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Cuisinart</td>
			<td>CPC-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-a-Lyzer Dialysis cassettes, 10K MWCO, 3 ml</td>
			<td>ThermoFisher</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Research Products International (RPI)</td>
			<td>S23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma-Aldrich</td>
			<td>1064461000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic (Na2HPO4)</td>
			<td>Research Products International (RPI)</td>
			<td>S23100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Research Products International (RPI)</td>
			<td>S24065</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Research Products International (RPI)</td>
			<td>T60040</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered Saline</td>
			<td>Boston Bio-Products</td>
			<td></td>
			<td>25mM Tris, 2.7mM KCl, 137 mM NaCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Research Products International (RPI)</td>
			<td>T60050</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Red</td>
			<td>Vector Labs</td>
			<td>SK-5105</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct detection of isolevuglandins in tissues using a d11 scfv-alkaline phosphatase fusion protein and immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

In representative experiments, D11 scfv with an alkaline phosphatase conjugation (D11-AP) was used in immunofluorescence to detect IsoLGs present in angiotensin II-treated mice compared to normal sham mice and humans with hypertension compared to normotensive humans. Mice were treated with angiotensin II at a dose of 490 ng/kg/min for two weeks, and hypertension was confirmed with increased systolic blood pressure compared to sham mice10. To ensure specificity of D11-AP, tissues were stained with or without the presence of D11-AP. As demonstrated by D11-AP staining, the aorta of mice with angiotensin II-induced hypertension showed elevated concentration of IsoLGs when compared to control mice (Figure 1). Background staining or autofluorescence was limited, as shown by negative controls that were not stained with D11-AP.
D11-AP was used to detect IsoLGs present in intestinal tissues of human patients with hypertension (HTN) or normotensive humans (NTN). Hypertension status was established from the hospital records as systolic blood pressure above 140 and diastolic blood pressure above 80 mmHg. Researchers developing immunofluorescence protocol for D11-AP were blinded to the hypertension status of human tissues. Sections were stained in the presence and absence of D11-AP to ensure antibody specificity and show background staining or autofluorescence. As shown in Figure 2, we found that tissues from patients with HTN had elevated concentrations of IsoLGs compared to patients with NTN. Staining without D11-AP also showed minimal background staining and autofluorescence. Endogenous alkaline phosphatase is expressed by intestinal epithelium, so the limited fluorescence of tissues stained without D11-AP shows the antigen retrieval used in this protocol was sufficient in inactivating endogenous alkaline phosphatase present in tissues. In combination with the results in mice, these results also show that the immunofluorescence protocol effectively shows elevated IsoLGs in hypertension when compared to normotensive status.
D11-AP was isolated and stored in the bacterial periplasmic extract. Mouse and human tissues were stained with D11-AP and periplasmic extract containing the AP linker without D11 to ensure that other factors that may be present in the periplasmic extract, such as excess or non-conjugated alkaline phosphatase, do not contribute to the staining observed in tissues treated with D11-AP (Figure 3). Tissues stained with D11-AP resulted in brighter staining compared to tissues stained with periplasmic extract. These results confirm that D11-AP is staining the tissues, and the staining is not due to non-conjugated bacterial alkaline phosphatase that can potentially be present in the periplasmic extract and result in false staining of IsoLGs or contribute to background staining.
A competitive control was performed by pre-incubating D11-AP with IsoLG adducted to MSA or non-adducted MSA before staining tissues to show the specificity of D11-AP to IsoLG. If D11-AP is specific for IsoLG, the antibody would bind to IsoLG-MSA, resulting in a depleted availability of D11-AP to stain tissues, and D11-AP incubated with non-adducted MSA would have similar staining to normal D11-AP. In tissues stained with D11-AP pre-incubated with the IsoLG competitor, we found diminished staining compared to tissues that were stained with D11-AP without any preincubation (Figure 4). In tissues stained with D11-AP preincubated with non-adducted MSA, we found staining to be similar to staining observed in tissues with D11-AP. These results show the specificity of D11-AP to IsoLGs due to reduced staining of tissues when D11-AP was pre-incubated with IsoLG/MSA, but not non-adducted MSA. In the final negative control, mouse tissue was stained with D11-AP or irrelevant scFv antibody, D20. Staining mouse aorta with D11-AP resulted in strong staining compared to D20 indicating the specificity of D11-AP to IsoLGs in hypertensive aortae (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62603/62603fig01.jpg" /> 
Figure 1: Immunofluorescence of the aorta in hypertensive and normotensive mice. Arteries from angiotensin II and sham infused mice were stained with and without D11-AP (D11) to show presence of IsoLGs. (A) Artery from an angiotensin II treated mouse probed with D11-AP (green) and nuclear counterstain (magenta), (B) Artery from a control sham treated mouse probed with D11-AP, (C) Artery from an angiotensin II treated mouse without D11-AP, (D) Artery from a control sham treated mouse without D11-AP (scale bar =100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62603/62603fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62603/62603fig02.jpg" /> 
Figure 2: Immunofluorescence of human intestinal tissues from hypertensive and normotensive patients. Tissues from patients with hypertension (HTN) and normotensive humans (NTN) were stained with and without D11-AP (D11) to show presence of IsoLGs in patients with HTN. (A) HTN tissues stained with D11-AP (green) and nuclear counterstain (magenta), (B) NTN tissues stained with D11-AP, (C) HTN tissues stained without D11-AP, (D) NTN tissues stained without D11-AP (scale bar =100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62603/62603fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62603/62603fig03.jpg" /> 
Figure 3: Mouse and human tissues stained with periplasmic extract with and without D11-AP. Mouse and human tissues were stained with periplasmic extracts with and without D11-AP. Images show limited staining of tissues with periplasmic extract without D11-AP, which show staining is mostly due to D11-AP binding rather than another component that may be present in periplasmic extract. (A) Ang mouse aorta stained with D11-AP (green) and nuclear counterstain (magenta), (B) Ang mouse aorta stained with periplasmic extract, (C) Sham mouse aorta stained with D11-AP, (D) Sham mouse aorta stained with periplasmic extract, (E) hypertensive human intestinal tissue stained with D11-AP, (F) hypertensive human intestinal tissue stained with periplasmic extract, (G) normotensive human intestinal tissue stained with D11-AP, (H) normotensive human intestinal tissue stained with periplasmic extract&#160;(scale bar =100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62603/62603fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62603/62603fig04.jpg" /> 
Figure 4: Competitive control in vessels of Ang and Sham mice. In this competitive control, D11-AP was pre-incubated with IsoLG-adducted MSA or non-adducted MSA. D11-AP without any pre-incubation was used as a control. These results show the specificity of D11-AP to IsoLGs because there is reduced staining of tissues with the IsoLG-MSA competitor compared to D11-AP. This reduction is due to IsoLG and not MSA because the non-adducted MSA pre-incubation resulted in staining similar to D11-AP control. Angiotensin II (A) and Sham (B) mouse aortae stained with D11-AP (green) and nuclear counterstain (magenta), Angiotensin II (C) and Sham (D) mouse aortae stained with D11-AP after incubating with IsoLG-MSA, Angiotensin II (E) and Sham (F) mouse aortae stained with D11-AP after incubating with non-adducted MSA (scale bar =100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62603/62603fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62603/62603fig05.jpg" /> 
Figure 5: Mouse aortae stained with D11 and irrelevant scFv, D20. Mouse tissue were stained with D11-AP and compared to an irrelevant control antibody, D20, which is specific for glycoprotein A2. Staining of tissue with D11-AP (green) and nuclear counterstain (magenta) (A) resulted in intense immunofluorescence compared to D20 (green) and nuclear counterstain (magenta) (B) which indicates specificity of D11-AP to IsoLGs (scale bar =100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62603/62603fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence at JoVE.com

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...

direct-detection-isolevuglandins-tissues-using-d11-scfv-alkaline

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1.9K Views.  Vanderbilt University Medical Center. This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples. 

Video: Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...