This study has been supported by the Department of Biochemistry, Sri Venkateswara College, University of Delhi. The authors thank the laboratory staff for their support.

The entire protocol with all steps is depicted as a schematic diagram in Figure 4. The method is adapted from a previous study using rat intestines8. The experiment was performed in compliance with institutional guidelines. The samples used in this study were procured from a commercial vendor.
CAUTION: Wear gloves during this experiment.
1. Preparation of inverted Jejunum sacs
<ol>
	<li>Pre-treat and clean goat jejunum.
	<ol>
		<li>Procure the entrails of a freshly sacrificed healthy goat from a licensed vendor.</li>
		<li>Place the entrails in enough 1x phosphate buffer saline (PBS) so that they are immersed completely for cleaning.</li>
		<li>Separate the small intestine from the large intestine, and make sure that the external connective tissue is removed using scissors.</li>
		<li>Gently flush the small intestine with 1x PBS using a 10 mL syringe, ensuring the removal of undigested food material to obtain a hollow bag of small intestine. Repeat this process at least thrice for effective cleaning. 
		NOTE: Avoid using excessive force or needles while flushing. The use of force can result in the puncturing of the intestinal walls and thus damage the intestinal enterocytes.</li>
		<li>Measure the complete length of the small intestine using a ruler to obtain the three parts: Duodenum (1/4th of the small intestine) and the remainder split evenly into Jejunum and Ileum.</li>
		<li>Proceed with the experiment using the Jejunum part.</li>
	</ol>
	</li>
	<li>Prepare inverted Jejunum.
	<ol>
		<li>Clean the jejunum part by removing all the connective tissue using a blade.</li>
		<li>Cut the jejunum into 12-15 cm long portions, wash with 1x PBS, and place in a Petri dish containing PBS.</li>
		<li>Invert the jejunum portions using a glass rod to allow the villi portion to be exposed on the outside. 
		CAUTION: Avoid using a glass rod with sharp edges. Moreover, the diameter of the glass rod should not be greater than the diameter of the sac. 
		NOTE: At this point, one can visualize the villi under a light microscope (Figure 4).</li>
		<li>Gently flush the inverted jejunum portions with 1x PBS.</li>
		<li>Check for any leaks from the sides of the inverted jejunum.</li>
		<li>Cut the inverted jejunum portions into 5 cm long sacs.</li>
		<li>Tie one end with twine and recheck for visual leaks at the tied end by filling the sacs with PBS.</li>
		<li>Tie the other end to create an empty inverted sac with villi on the external surface.</li>
		<li>Recheck for external leaks by patting the inverted sac on filter paper.</li>
	</ol>
	</li>
	<li>Equilibrate the inverted sacs in 1x PBS.
	<ol>
		<li>Proceed with the experiment on the same day or store the sacs overnight in 1x PBS at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Experimental setup for Histidine transport
<ol>
	<li>Assemble two sets of three 50 mL tubes according to different concentrations of sodium chloride as given in Table 1.</li>
	<li>Dip the prepared intestinal sacs in the specified tubes for 30 min and 60 min for each of the sets.</li>
	<li>After the designated time, empty the solution inside the sac in a separate 1.5 mL microcentrifuge tube and determine the volume.</li>
	<li>Aspirate the liquid inside the sac using a 2 mL syringe. Alternatively, untie one end of the sac and collect the contents in a fresh 1.5 mL tube.</li>
	<li>Assay 0.1 mL of the collected samples for histidine estimation and calculate the concentration using the standard graph.</li>
</ol>
3. Estimation of Histidine using Pauly&#39;s reaction
<ol>
	<li>Prepare a standard curve for histidine.
	<ol>
		<li>Prepare solutions with varying concentrations of histidine as indicated in Table 2 using stock solutions of histidine (5 mM) and water.</li>
		<li>Add sulphanilic acid (0.5 mL) and sodium nitrate (0.5 mL) to each tube.</li>
		<li>Incubate the tubes for 5 min at room temperature, then add 1 mL each of sodium carbonate and ethanol to each tube.</li>
		<li>Incubate the tubes for 20 min at room temperature (~20 &#176;C) and note the absorbance at wavelength 490 nm.</li>
		<li>Plot a standard curve of absorbance versus Histidine concentration. 
		NOTE: This test for histidine is based on the property of histidine to form a diazo compound (Figure 5).</li>
	</ol>
	</li>
</ol>

Sodium-Dependent Histidine Absorption in Goat Jejunal Inverted Sacs

<ol>
	<li>Nelson, D. L., Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lehninger Principles of Biochemistry. 7th Edition. , (2017).</li><li>Stillwell, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Membrane Transport. An Introduction to Biological Membranes. , (2013).</li><li>Fick, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+liquid+diffusion.">On liquid diffusion.</a> The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science. 10 (63), 30-39 (1855).</li><li>Sherwood, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Human Physiology. , (2013).</li><li>Br&#246;er, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+amino+acid+transport+and+metabolic+health.">Intestinal amino acid transport and metabolic health.</a> Annu Rev Nutr. 43, 73-99 (2023).</li><li>Avissar, N. E., Ryan, C. K., Ganapathy, V., Sax, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+-dependent+neutral+amino+acid+transporter+ATB0+is+a+rabbit+epithelial+cell+brush-border+protein.">Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial cell brush-border protein.</a> Am J Physiol Cell Physiol. 281 (3), C963-C971 (2001).</li><li>Br&#246;er, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+transport+across+mammalian+intestinal+and+renal+epithelia.">Amino acid transport across mammalian intestinal and renal epithelia.</a> Physiol Rev. 88 (1), 249-286 (2008).</li><li>Agar, W. T., Hird, F. J. R., Sidhu, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uptake+of+amino+acids+by+the+intestine.">The uptake of amino acids by the intestine.</a> BBA - Biochim Biophys Acta. 14 (1), 80-84 (1954).</li><li>Pauly, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Konstitution+des+Histidins.">&#220;ber die Konstitution des Histidins.</a> I. Mitteilung. Biological Chemistry. 42 (5-6), 508-518 (1904).</li><li>Wilson, T. H., Wiseman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+sacs+of+everted+small+intestine+for+the+study+of+the+transference+of+substances+from+the+mucosal+to+the+serosal+surface.">The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface.</a> J Physiol. 123 (1), 116-125 (1954).</li><li>Barthe, L., Woodley, J. F., Kenworthy, S., Houin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+everted+gut+sac+as+a+simple+and+accurate+technique+to+measure+paracellular+transport+across+the+small+intestine.">An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine.</a> Eur J Drug Metab Pharmacokinet. 23 (2), 313-323 (1998).</li><li>Alam, M. A., Al-Jenoobi, F. I., Al-Mohizea, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everted+gut+sac+model+as+a+tool+in+pharmaceutical+research:+Limitations+and+applications.">Everted gut sac model as a tool in pharmaceutical research: Limitations and applications.</a> J Pharm Pharmacol. 64 (3), 326-336 (2012).</li><li>Pento, J. T., Mousissian, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+deterioration+of+active+transport+in+duodenal+segments+of+rat+intestine.">Time-dependent deterioration of active transport in duodenal segments of rat intestine.</a> J Pharmacol Methods. 20 (1), 9-14 (1988).</li><li>Williams, L., Sembiante, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiential+learning+in+U.S.+undergraduate+teacher+preparation+programs:+A+review+of+the+literature.">Experiential learning in U.S. undergraduate teacher preparation programs: A review of the literature.</a> Teach Teach Educ. 112, 103630 (2022).</li></ol>

The authors have no competing financial interests or other conflicts of interest.

Membrane transport is one of the most fundamental concepts taught to undergraduate students of all major biological science disciplines, basic or applied. Traditionally, movement across membranes has been visualized using metabolites labeled with radioactive isotopes. However, these methods are extremely hazardous and not feasible for teaching or learning. While experiential learning is the best pedagogical technique to understand such complex concepts, it is a challenge that is further compounded by the lack of infrastr...

Biological cells are surrounded by a membrane lipid bilayer that separates the intracellular cytosol from the extracellular content. The membrane serves as a semipermeable barrier that regulates the movement of solutes1. Transport across biological membranes is affected by a solute&#39;s permeability coefficient, which depends on several factors, including the concentration and charge of the solute. In general, solutes move through the membrane using three mechanisms (Figure 1): Passive diffusion, facilitated diffusion, and active transport2. Simple diffusion is the process by which soluble, uncharged, and non-polar solutes pass down their concentration gradient through a semipermeable membrane (Figure 1A). Membrane proteins do not assist in this process since it involves the movement of solutes from a region of higher concentration to a region of lower concentration. The rate of diffusion is based on Fick&#39;s Law3. On the other hand, facilitated diffusion is a protein-dependent transport wherein the membrane allows only selective solutes to pass along a concentration gradient without the expenditure of energy (Figure 1B). This kind of transport is specific and differs from simple diffusion in exhibiting saturation kinetics.
Active transport is a protein-dependent transport of molecules against their concentration gradient, i.e., from the region of lower concentration to a region of higher concentration with the use of ATP or ion gradients (Figure 1C). When the transporter hydrolyses ATP, the transport is termed primary active transport (Figure 1C; left panel). Another form of active transport is secondary active transport (Figure 1C; right panel). In secondary active transport, solutes are moved based on an electrochemical gradient. It takes place when a transporter protein couples the movement of an ion (typically Na+) down its concentration gradient with the movement of another molecule or an ion against its concentration gradient. This kind of solute movement can be a co-transport (Symport) wherein both the solute and the ion move in the same direction or an exchange (Antiport), in which case the solute and the ion move in opposite directions.
The dietary amino acids and monosaccharides from food sources are absorbed in the small intestine. The small intestine can be functionally divided into three segments: Duodenum, Jejunum, and Ileum (Figure 2). Absorption of solute occurs throughout the small intestine, with maximum absorption occurring at the jejunum and at the proximal end of the ileum. The intestinal enterocytes are polarized cells, and the tight junctions connecting two adjacent cells create two distinct membrane sites - the basolateral and the apical membrane site (Figure 2). The absorption of luminal solutes generated by digestion occurs on the apical membrane site4.
Histidine transport in the intestinal enterocytes at the apical membrane is an example of a secondary active symport that is sodium-dependent. At the basolateral end, the histidine entering the enterocyte moves down the concentration gradient into the hepatic portal circulation. The intracellular concentrations of sodium within the enterocyte are maintained at 12 mmoles/L1, which is lower than the extracellular/luminal concentrations, due to the active pumping of sodium out of the cell by Na+ K+ATPase located on the basolateral membrane (Figure 3). At the apical membrane of enterocytes, the B0AT and the sodium neutral amino acid transporter (SNAT) 5 are the major transporters that not only transport histidine but also amino acids such as asparagine and glutamine in a sodium-dependent cotransport5,6. Another transport protein called Large Amino acid Transporter (LAT)1 present at the basolateral membrane of enterocytes transports large neutral amino acids such as leucine, tryptophan, tyrosine, and phenylalanine across the membrane7.
With the objective of teaching the concept of membrane transport integrated with techniques such as spectrophotometry and routine biochemical assays through experiential learning pedagogy, it is imperative to develop methodologies that can not only demonstrate the concept in easy-to-understand terms but also enable participatory learning for undergraduate students. Currently, there are limited resources available to the students for hands-on engagement to learn such concepts of biochemistry. Here, we report a simple protocol for demonstrating Histidine transport across goat intestinal membrane that is easy to reproduce in undergraduate laboratories and can be adapted for evaluating the transport of other metabolites as well. More importantly, the method utilizes inexpensive materials in an undergraduate laboratory, thereby enabling experiential learning in even the simplest of laboratory settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>TARSONS</td>
			<td>500020</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Test Tubes</td>
			<td>BOROSIL</td>
			<td>9800U04</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Sterile Falcon Tubes</td>
			<td>TARSONS</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Beaker</td>
			<td>BOROSIL</td>
			<td>10044977</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Conical Flask</td>
			<td>BOROSIL</td>
			<td>691467</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SRL</td>
			<td>42738</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Spectrophotometer</td>
			<td>SYSTRONICS</td>
			<td>2710</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>1009831000</td>
			<td></td>
		</tr>
		<tr>
			<td>Finpipettes</td>
			<td>THERMOFISHER</td>
			<td>4642090</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Stirrer Rod</td>
			<td>BOROSIL</td>
			<td>9850107</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine&#160;</td>
			<td>SRL</td>
			<td>17849</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SRL</td>
			<td>41721</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>KIMTECH</td>
			<td>112-4847</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish&#160;</td>
			<td>TARSONS</td>
			<td>460090</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (ph 7.4)</td>
			<td>SRL</td>
			<td>95131</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>ABDOS</td>
			<td>P10102</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>SRL</td>
			<td>89382</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrate&#160;</td>
			<td>SRL</td>
			<td>44618</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic (anhydrous)</td>
			<td>SRL</td>
			<td>53046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic (anhydrous)</td>
			<td>SRL</td>
			<td>22249</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulphanilic Acid&#160;</td>
			<td>SRL</td>
			<td>15354</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of membrane transport of histidine using goat intestinal inverted sacs: an experiential pedagogical tool for undergraduates

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular circulation. Absorption of dietary histidine relies largely on the sodium-coupled neutral amino acid transport by the Broad neutral amino acid transporter (B0AT) present on the apical membrane of the enterocyte. Here, we demonstrate the absorption of histidine by the intestinal villus enterocytes from the lumen using goat jejunal inverted sacs. The jejunal sacs exposed to varying concentrations of sodium and histidine were assayed to determine the concentration of histidine inside the sacs as a function of time. The results show active histidine absorption. Increasing the concentration of salt resulted in higher absorption of histidine, suggesting a symport of sodium and histidine absorption in goat intestinal inverted sacs. This protocol may be applied to visualize the intestinal mobility of amino acids or other metabolites with appropriate modifications. We propose this experiment as an experiential pedagogical tool that can help undergraduate students comprehend the concept of membrane transport.

The experimental workflow for demonstrating intestinal mobility of histidine through absorption of histidine by intestinal villi into the lumen of the inverted sacs is illustrated in Figure 4, Table 1 and Table 2. Three independent experimental setups were performed, and representative data are presented in Figure 6.
Under the given experimental conditions, Histidine estimation using Pauly&#39;s react...

Author Spotlight: Experiential Tool for Teaching Active Transport Using Ex Vivo Histidine Uptake

Here, we report an inexpensive and reproducible method demonstrating the membrane transport of histidine in a goat intestine. This process occurs by co-transporting histidine and sodium ions enabled by the sodium gradient across the enterocyte membrane. This method exploits experiential learning pedagogy to better understand solute movement across biological membranes.

Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular ...

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Biochemistry

1.4K Views.  University of Delhi. Here, we report an inexpensive and reproducible method demonstrating the membrane transport of histidine in a goat intestine. This process occurs by co-transporting histidine and sodium ions enabled by the sodium gradient across the enterocyte membrane. This method exploits experiential learning pedagogy to better understand solute movement across biological membranes.

Video: Author Spotlight: Experiential Tool for Teaching Active Transport Using Ex Vivo Histidine Uptake

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...