This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

1. Cell Culture
<ol>
	<li>Use human telomerase reverse transcriptase (hTERT) immortalized retinal pigment epithelial cells (hTERT-RPE1; referred to here as RPE1). 
	NOTE: RPE1 cells are near-diploid, non-transformed human cells that are commonly used to study centriole assembly and ciliogenesis. These cells follow a normal centriole duplication cycle coordinated with a regulated cell cycle.</li>
	<li>Passage a near-confluent 100 mm cell culture dish of RPE1 cells at 1:5 dilution of the original culture into a fresh 100 mm dish containing 10 ml of DME/F-12 (1:1) medium supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin G and 100 &#181;g/ml streptomycin (the complete medium is referred as DME/F-12 medium). Grow cells at 37 &#176;C in the presence of 5% CO2.
	<ol>
		<li>Incubate 12 mm round glass coverslips in 50 ml of 1 N HCl in a covered glass beaker, O/N without shaking, at 60 &#176;C in a water bath or incubator.</li>
		<li>After discarding the acid solution, wash the coverslips three times in 100 ml distilled water by incubating for 15 min with occasional shaking. Repeat the washing similarly in 70% Ethanol and 95% Ethanol.</li>
		<li>Dry the coverslips by individually spreading them out on a laboratory blotting paper in a biosafety cabinet, followed by sterilization with UV radiation for 60 min.</li>
	</ol>
	</li>
	<li>Place 2-4 dry coverslips in a 35 mm cell culture dish. Dilute the solution of fibronectin or fibronectin like engineered polymer (stock concentration of 1 mg/ml) to a concentration of 25 &#181;g/ml in cell culture PBS.
	<ol>
		<li>Place 80-100 &#181;l of the diluted solution onto each coverslip and incubate for 60 min to coat the top side of the coverslip. Wash the coverslips thrice using PBS before adding complete medium.</li>
	</ol>
	</li>
</ol>
2. Growing Cells and Treating Cells with Proteasome Inhibitors
<ol>
	<li>Passage 2 x 105 asynchronously growing RPE1 cells in each 35 mm dish containing coverslips and grow the cells in 2 ml complete medium. Replace the culture medium every 24 hr with fresh pre-warmed complete medium.
	<ol>
		<li>To analyze the effect of proteasome inhibition on the centrosomal level of the test protein (here VDAC3 or &#947;-tubulin), grow the cells in two 35 mm dishes for 44 hr. Replace the culture medium with complete medium and add either MG115 or DMSO as the control solvent at a final concentration of 5 &#181;M or 0.05% respectively. At the same time, add BrdU to the cells at a final concentration of 40 &#181;M and incubate cells for 4 hr.</li>
		<li>Transfer each coverslip in a 24-well plate. Add 500 &#181;l chilled methanol to each well and incubate the plate at -20 &#176;C for 10 min to fix the cells on the coverslips. Immediately wash the coverslips three times with 500 &#181;l wash buffer (1x PBS containing 0.5 mM MgCl2 and 0.05% Triton-X 100).</li>
	</ol>
	</li>
	<li>Harvest the residual cells from the 35 mm dish and centrifuge the cells at 1,000 x g for 5 min. Use the cell pellet to analyze the total protein by western blotting (step 6).</li>
</ol>
3. BrdU Pulse and Chase Assay to Analyze Protein Levels in Different Cell Cycle Phases
<ol>
	<li>To analyze the variation of the level of a protein (here VDAC3, Sas6 or Cep135) at centrosomes at different phases of the cell cycle, grow cells in two 35 mm dishes containing coverslips for 44 hr. Replace the culture medium with complete medium containing 40 &#181;M BrdU and incubate the cells for 4 hr in the cell culture incubator.</li>
	<li>From one dish, transfer the coverslips to a 24-well plate to fix the cells using chilled methanol as described in step 2.1.2.</li>
	<li>After the 4 hr BrdU pulse, remove the BrdU-containing medium, wash the cells once with PBS and once with complete medium, add fresh medium, and then grow the cells in the absence of BrdU for various times (typically another 4 hr for RPE1 cells) before fixing the cells as described in step 2.1.2. 
	NOTE: For RPE1 cells, the majority of the BrdU-positive cells are in late S or G2-phase after a 4 hr chase. This must be determined independently for each cell type.</li>
</ol>
4. Immunostaining
<ol>
	<li>Incubate the fixed cells on coverslips in 200 &#181;l blocking buffer (2% BSA and 0.1% TritonX-100 in 1x PBS) for 30 min.
	<ol>
		<li>Incubate the cells with a mix of primary antibodies (typically one raised in rabbit and another raised in mouse) diluted in blocking buffer O/N at 4 &#176;C in a humidified chamber.</li>
		<li>To make a humidified chamber, put a wet paper towel in the bottom half of an empty 1,000 &#181;l pipette tip box. Lay a strip of Parafilm on the rack surface, and spot a droplet (15-20 &#181;l) of the antibody solution onto the Parafilm for each coverslip to be incubated. Invert a coverslip onto a droplet of antibody solution (so that cells are immersed) and close the lid of the tip box.</li>
		<li>Invert coverslips again and return them back to the 24-well dish. Wash coverslips and then incubate them in 150 &#181;l secondary antibody mixture (here green-fluorescent dye conjugated anti-rabbit and red-fluorescent dye conjugated anti-mouse diluted in blocking buffer) for 1 hr at RT. Wash the coverslips three times. 
		NOTE: The fluorophore-conjugated Secondary antibodies are light sensitive. Protect the samples from light during steps 4.1.3-5.1.2.</li>
	</ol>
	</li>
	<li>Prepare for anti-BrdU staining by fixing the stained RPE1 cells again with 500 &#181;l chilled methanol at -20 &#176;C for 10 min, as described in step 2.1.2. Wash the coverslips thrice. 
	NOTE: This fixation secures the primary and secondary antibody labeling of the protein(s) of interest during the acid hydrolysis process in the following step.
	<ol>
		<li>Incubate the cells in 200 &#181;l of 2 N HCl for 30 min at RT. Neutralize with 300 &#181;l of 1 M Tris-Cl, pH 8 and wash the cells three times using wash buffer.</li>
		<li>Block the cells again using 200 &#181;l blocking buffer for 30 min at RT.</li>
		<li>Incubate the cells with rat anti-BrdU antibody (diluted in blocking buffer) for 45 min at 37 &#176;C in a humidified chamber. Return the coverslips back to the 24-well dish, wash them and then incubate with blue-fluorescent dye conjugated anti-rat secondary antibody (diluted in 150 &#181;l blocking buffer in 24-well dish for 1 hr at RT.</li>
	</ol>
	</li>
	<li>Wash the coverslips three times. Spot a droplet (roughly 3-6 &#181;l) of a mounting solution containing antifade reagent on a glass microscope slide. Invert a coverslip, with the cell-side facing down, onto the mounting solution. Wipe excess liquid by gently pressing the coverslip against the slide using soft cleaning tissue. Apply transparent nail polish along the edge of the coverslip to seal it onto the microscope slide.</li>
</ol>
5. Immunofluorescence Image Acquisition and Analysis
<ol>
	<li>Use a 100X Plan Apo oil immersion objective (with a 1.4 numerical aperture) to acquire the images of BrdU-positive RPE1 cells at ambient temperature.
	<ol>
		<li>Put a slide on the microscope (see equipment) attached with a camera capable of digital imaging. For each fluorophore, determine the appropriate exposure time (typically between 300-1,000 msec) by manually examining all samples of an experiment. Before acquiring images of a cell, manually determine the appropriate top and bottom focal plane along the Z-axis (typically 0.2 &#181;m step size). Acquire images of different samples that are on separate coverslips using identical exposure time for different fluorophores along the Z-axis using a digital microscopy imaging software package.</li>
	</ol>
	</li>
	<li>Perform deconvolution (in these cases using No neighbor algorithm) of all image stacks acquired along the Z-axis.
	<ol>
		<li>Obtain the total intensity projection of each image stack along the Z-axis.</li>
	</ol>
	</li>
	<li>In cells where centrosomes are close (distance between two centrosomes is less than 2 &#181;m), draw a small square (typically 20-30 pixels per side) around both centrosomes and mark the selected area.
	<ol>
		<li>Draw a larger square (typically 24-35 pixels per side) surrounding the first square and mark the selected area of the large square.</li>
		<li>Obtain the area (A) and the total fluorescence intensity (F) of each fluorophore in each box (S denotes small box and L denotes large box).</li>
		<li>Analyze the background corrected fluorescence intensity of each fluorophore using the formula described by Howell et al.29: F= FS &#8211; [( FL&#8211; FS) x AS /( AL &#8211; AS)].</li>
		<li>Obtain the normalized fluorescence intensity of the protein of interest (here VDAC3 or Sas6) by calculating the ratio of the background-corrected fluorescence intensity of its fluorophore to that of the fluorophore used for the chosen internal standard (here &#947;-tubulin, Sas6 or Cep135).</li>
	</ol>
	</li>
	<li>In the case of analyzing cells where centrosomes are well separated (distance between two centrosomes is greater than 2 &#181;m), analyze each centrosome separately by drawing two separate sets of boxes. Draw a small square (typically 15-25 pixels per side) around each centrosome and mark the selected area. Draw a larger square (20-30 pixels per side) surrounding the first square and mark the selected area.
	<ol>
		<li>Obtain the area (A) and the total fluorescence intensity (F) of each fluorophore in each box, and calculate the background corrected fluorescence intensities of each fluorophore, separately for each centrosome, as described in step 5.3.3.</li>
		<li>Combine the background corrected fluorescence intensities of each protein from the two centrosomes to obtain the total centrosomal level of that protein in that cell.</li>
		<li>Obtain the normalized intensity for the protein of interest by calculating the ratio of its total centrosomal intensity to the total centrosomal intensity of the internal standard.</li>
	</ol>
	</li>
	<li>Analyze at least 15-25 cells for each experimental condition and plot the graph in a spreadsheet.</li>
</ol>
6. Analyzing the Total Protein Using Western Blotting
<ol>
	<li>Resuspend the cells in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and incubate for 10 min on ice to lyse the cells.
	<ol>
		<li>Centrifuge the mixture at 10,000 x g for 10 min, separate the lysate from the pellet fraction and measure the total protein concentration of each lysate using a bicinchoninic acid (BCA) assay kit.</li>
	</ol>
	</li>
	<li>Mix 40 &#181;g of lysate with 4x SDS-PAGE loading buffer (50 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 100 mM Dithiothreitol, 0.1% Bromophenol blue).
	<ol>
		<li>Boil the samples for 10 min and run the samples on a 12.5% denaturing SDS-PAGE at a constant voltage of 200 V.</li>
	</ol>
	</li>
	<li>Transfer the proteins separated on SDS-PAGE onto a nitrocellulose membrane using western blotting technique (at a constant voltage of 90 V for 1 hr in cold).
	<ol>
		<li>Block the membrane with 3% non-fat milk in 1x PBS, and then incubate the membrane with solutions of diluted primary antibodies (in this case rabbit anti-VDAC3, rabbit anti-&#947;-tubulin and mouse anti-&#945;-tubulin) for 1 hr at RT.</li>
		<li>After washing the membrane three times (each 5 min incubation under shaking) using PBS-T buffer (1x PBS and 0.2% Tween-20), incubate the membrane in a mixture of near-Infrared (IR) fluorescent dye conjugated anti-rabbit and IR fluorescent dye conjugated anti-mouse secondary antibodies (diluted in PBS-T) for 1 hr.</li>
		<li>Wash the membrane three times and then scan the membrane to analyze the bands using an infrared fluorescence imaging system suitable for immunoblot detection.</li>
	</ol>
	</li>
</ol>

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J. 32, 1141-1154 (2013).</li><li>Lichtenstein, N., Geiger, B., Kam, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cytoskeletal+organization+by+digital+fluorescent+microscopy.">Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.</a> Cytometry A. 54, 8-18 (2003).</li><li>Keller, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+the+centriole+proteome:+the+conserved+WD40+domain+protein+POC1+is+required+for+centriole+duplication+and+length+control.">Molecular architecture of the centriole proteome: the conserved WD40 domain protein POC1 is required for centriole duplication and length control.</a> Mol. Biol. Cell. 20, 1150-1166 (2009).</li><li>Venoux, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poc1A+and+Poc1B+act+together+in+human+cells+to+ensure+centriole+integrity.">Poc1A and Poc1B act together in human cells to ensure centriole integrity.</a> J. Cell Sci. 126, 163-175 (2013).</li><li>Stevens, N. R., Roque, H., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSas-6+and+Ana2+coassemble+into+tubules+to+promote+centriole+duplication+and+engagement.">DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement.</a> Dev. Cell. 19, 913-919 (2010).</li><li>Machiels, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+analysis+of+cell+cycle+kinetics+upon+proteasome+inhibition.">Detailed analysis of cell cycle kinetics upon proteasome inhibition.</a> Cytometry. 28, 243-252 (1997).</li><li>Yang, C. H., Kasbek, C., Majumder, S., Yusof, A. M., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mps1+phosphorylation+sites+regulate+the+function+of+centrin+2+in+centriole+assembly.">Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly.</a> Mol. Biol. Cell. 21, 4361-4372 (2010).</li><li>Tsou, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polo+kinase+and+separase+regulate+the+mitotic+licensing+of+centriole+duplication+in+human+cells.">Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells.</a> Dev. Cell. 17, 344-354 (2009).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Buck, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+S-phase+cell+cycle+progression+using+5-ethynyl-2'-deoxyuridine+incorporation+with+click+chemistry,+an+alternative+to+using+5-bromo-2'-deoxyuridine+antibodies.">Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.</a> Biotechniques. 44, 927-929 (2008).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> J. Cell Biol. 185, 1135-1148 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Quantitative microscopy in cell biology is commonly associated with live-cell imaging assays such as Fluorescence Resonance Energy Transfer (FRET), Fluorescence Recovery After Photobleaching (FRAP), etc. However, there are growing examples of cell biologists developing different quantitative microscopy assays for fixed cells in recent years27,34-36. Importantly, progress in understanding centrosome biology often requires understanding of the centrosome-specific function of proteins whose centrosomal p...

Centrosomes consist of a pair of centrioles surrounded by pericentriolar material (PCM). Being the major microtubule organizing centers (MTOCs) in mammalian cells, centrosomes serve as the two poles of mitotic spindles in dividing cells, and thus help maintain genomic integrity1. In quiescent cells (e.g., during G0 phase), one of the two centrioles of the centrosome, namely the mother centriole, is transformed into a basal body to assemble the primary cilium, a sensory organelle protruding out from the cell surface2. Once the cells re-enter the cell cycle, primary cilia are disassembled and each centriole directs the assembly of a procentriole at its proximal end that gradually elongates to form a mature centriole3. At the onset of S-phase, a cartwheel-like structure that provides the 9-fold symmetry to the centriole is formed on the surface of each existing centriole and will become the base of each procentriole. Sas6 that is indispensable for centriole assembly is recruited to form the hub of the cartwheel4-6. Other centriolar proteins are then assembled onto the cartwheel in a highly regulated, proximal to distal manner7. After precisely completing centriole duplication, cells assemble additional pericentriolar materials to build two functional centrosomes by the end of G2 phase8. In addition to the core centriolar components9-11, several other proteins including kinases, phosphatases, chaperones, scaffold components, membrane associated proteins and degradation machinery are associated with centrioles, basal bodies and PCM at different times of the cell cycle12-16. It is often noted that the centrosomal levels of many proteins are temporally regulated by centrosomal targeting mechanisms and/or proteasomal degradation at centrosomes. Importantly, the fluctuations in the centrosomal level of several proteins such as Plk4, Mps1, Sas6, and CP110 at different points of the cell cycle appears to be crucial to regulate centriole assembly5,17-22, and in the case of Mps1 preventing this centrosomal degradation leads to the formation of excess centrioles19. On the other hand, the centrosomal fractions of several proteins are less labile compared to cytosolic pools. For example siRNA-mediated down-regulation of Centrin 2 (Cetn2) led to only a moderate decrease of the protein level at the centrioles despite great reduction in its whole cell levels23. It is therefore crucial to measure the changes in the level of centrosomal proteins at the centrosome rather than measuring the whole cell protein levels when assessing their centrosome-specific functions.
In this study we have developed an assay using indirect immunofluorescence (IIF) to quantify the relative level of a protein at centrosomes. This assay is developed particularly to analyze cells that are from different samples and thus can not be imaged at the same time. These samples can be cells that were treated with different reagents (i.e., drug versus control), collected at different time points (i.e., pulse versus chase), or are in different phases of the cell cycle. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region and to normalize that value against the same for another protein whose levels do not vary under the chosen experimental conditions. Several studies in centrosome biology have recently utilized various quantitative microscopy techniques, in both live or fixed cells, to determine the centrosome-specific function of candidate proteins24-27. Similar to those assays, the present technique also measures the background corrected fluorescence intensity of the test protein. However, the inclusion of the normalization using an internal standard in this assay would likely offer greater accuracy and confidence in analyzing two different samples that are on two different coverslips. Moreover, in addition to examining the protein level at centrosomes, with minor adjustments this method can be applied to a diverse set of experimental conditions or at other cellular sites.
Here, we combine our quantitative microscopy assay with a BrdU pulse-chase strategy to compare cells from different cell cycle stages. Instead of using standard cell cycle arrest and release techniques to study various cell cycle time points, asynchronously growing cells are incubated with BrdU to label cells in S-phase, and the labeled cells are chased for various times (typically 4-6 hr). Most of the labeled cells will be in S-phase immediately after the pulse. The length of the chase is chosen so that after the chase, labeled cells will be in late S, G2, or mitosis, which can be verified by morphological characteristics such as- position of centrosomes with respect to nuclei, distance between centrosomes, condensation of chromosomes etc. Thus, the length of the chase depends on the average duration of S, G2 and M phase of a particular cell type. Since this approach avoids cell cycle inhibitors such as hydroxyurea, aphidicolin, nocodazole, etc., it allows a more physiologically relevant cell cycle analysis.
Thus, we demonstrate here that the quantitative fluorescence microscopy assay alone, or in combination with BrdU pulse-chase assay, is a simple yet powerful technique to accurately measure the relative changes in the centrosomal level of a candidate protein during an unperturbed cell cycle. We measured the centrosomal level of VDAC3, a protein that we recently identified at centrosomes in addition to mitochondria16,28, using these assays. Results obtained here verify our previous observation that the centrosomal pool of VDAC3 is regulated by degradation, and also varies in a cell cycle dependent manner16, furthermore validating the applicability of this method.

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			<td style="width:105px;">Company&#160;</td>
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			<td style="width:99px;">D2650</td>
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			<td height="20" style="height:20px;width:179px;">MG115</td>
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			<td style="width:195px;">Stock of 10 mM in DMSO</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:179px;">BrdU</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">B5002</td>
			<td style="width:195px;">Stock of 10 mM in DMSO</td>
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			<td height="60" style="height:60px;width:179px;">Anti-g-tubulin (mouse monoclonal, clone GTU 88)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T 6557</td>
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			<td style="width:195px;">1:100 in IIF blocking buffer</td>
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			<td height="40" style="height:40px;width:179px;">Anti-Cep135 (rabbit polyclonal)</td>
			<td style="width:105px;">Abcam</td>
			<td style="width:99px;">ab-75005</td>
			<td style="width:195px;">1:500 in IIF blocking buffer</td>
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			<td style="width:195px;">1:250 in IIF blocking buffer</td>
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			<td style="width:195px;">1:200 in IIF blocking buffer</td>
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			<td height="60" style="height:60px;width:179px;">Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21202</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21203</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
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		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21206</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21207</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-g-tubulin (rabbit polyclonal)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T5192</td>
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			<td height="40" style="height:40px;width:179px;">Anti-a-tubulin (mouse monoclonal, DM1A)</td>
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			<td style="width:195px;">1:20000 in WB blocking buffer</td>
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			<td height="40" style="height:40px;width:179px;">Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A10043</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
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			<td height="60" style="height:60px;width:179px;">Mouse IgG (H&#38;L) Antibody IRDye800CW Conjugated</td>
			<td style="width:105px;">Rockland antibodies</td>
			<td style="width:99px;">610-731-002</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">SlowFade Gold Antifade Reagent&#160;</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">S36936</td>
			<td style="width:195px;">Mounting media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Round coverslips 12CIR.-1</td>
			<td style="width:105px;">Fisherbrand</td>
			<td style="width:99px;">12-545-80</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Olympus IX-81 microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Retiga ExiFAST 1394 IR camera</td>
			<td style="width:105px;">QImaging&#160;</td>
			<td style="width:99px;">32-0082B-238</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">100X Plan Apo oil immersion objective</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;">1.4 numerical aperture</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Slidebook software package</td>
			<td style="width:105px;">Intelligent Imaging Innovations</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Odyssey IR Imaging System</td>
			<td style="width:105px;">Li-cor Biosciences</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Bicinchoninic acid (BCA) assay</td>
			<td style="width:105px;">Thermo Scientific</td>
			<td align="right">23227</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow UV cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M622</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Blue cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M643</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Green cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M663</td>
			<td>Filter</td>
		</tr>
	</tbody>
</table>

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.

Our recent studies identified novel centrosomal localization and function of VDAC3, one of the mitochondrial porins16,28. Immunostaining of several mammalian cells including RPE1 cells using a VDAC3-specific antibody showed prominent centrosomal staining and comparatively weak mitochondrial staining. We also showed that centrosomal VDAC3 is preferentially associated with the mother centriole, and the centrosomal pool of both the endogenous and ectopically expressed VDAC3 is regulated by degradation16</sup...

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels 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 ...

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JoVE Journal

Biology

14.9K Views.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

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

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

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

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Chapters in this video

Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

The MultiBac Protein Complex Production Platform at the EMBL

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Imaging Centrosomes in Fly Testes

Measurement of Heme Synthesis Levels in Mammalian Cells

In vitro Methylation Assay to Study Protein Arginine Methylation

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions