The authors thank Blanka Duvnjak for her exceptional technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (www.dfg.de) WO1811/2-1 to M.W. and QU280/3-1 to I.Q. The funder had no role in the study design, data collection, data analysis, decision to publish, and preparation of the manuscript.

Mice were obtained as an in-house breed from the local animal care facility or from Janvier Labs in France. The investigations were conducted according to the guidelines outlined in the Guide for Care and Use of Laboratory Animals (U.S. National Institutes of Health Publication No. 85-23, revised 1996). All animal experiments were performed in accordance with the relevant institutional approvals (state government LANUV reference number AZ:84-02.04. 2016.A435).
1. Preparation of Instruments, Solutions, and Equipment
<ol>
	<li>Prepare 1 L of phosphate buffered saline supplemented with 1 mM magnesium chloride (MgCl2) and 0.1 mM calcium chloride (CaCl2) (PBSCM) and filter it through a sterile filter.</li>
	<li>Prepare 5 mL of sterile PBSCM per mouse for perfusion and place it on ice.</li>
	<li>For each mouse, prepare 5 mL of sterile PBSCM supplemented with 0.5 mg/mL biotin.</li>
	<li>For each mouse, prepare 5 mL of sterile PBSCM and add 0.8 x 108 magnetic beads (e.g., 200 &#181;L of a 4 x 108 beads/mL solution, without pretreatment) for embolization of the glomeruli. Prepare this solution under the cell culture bench to keep the magnetic beads in the original tube sterile. Place this solution on ice.</li>
	<li>Prepare a quenching solution by adding 100 mM glycine to PBSCM (5 mL per mouse) and keep it on ice.</li>
	<li>Make a collagenase solution (0.378 U/mL collagenase A in sterile PBSCM). Per mouse, pipette 1 mL of the collagenase solution into a 2 mL tube and place it on ice.</li>
	<li>For washing, prepare sterile PBSCM (see step 1.1) in a 50 mL tube and place it on ice.</li>
	<li>For perfusion, use a syringe pump with a flow of 2.0 mL/min.</li>
	<li>Prepare a 10 mL syringe with a 21G needle. Place the tip of the needle in a 20-30 cm long catheter (inner diameter, ID = 0.58 mm). Connect the catheter (ID 0.58 mm) with a 10 cm short catheter (ID 0.28 mm) and cut the tip of the smaller catheter oblique for an easy insertion into the mouse aorta.</li>
	<li>For ligature procedures during the surgery, cut three 5-7 cm silk threads (4-0 to 6-0) per mouse.</li>
	<li>For the surgery, prepare 3 surgical clamps, 2 surgical scissors, 2 tweezers, 2 fine tweezers, 1 fine scissor, and swabs. Prepare the anesthesia (e.g., intraperitoneal anesthesia ketamine 100 mg/kg bodyweight and xylazine 5 mg/kg bodyweight).</li>
	<li>For isolation of the glomeruli of two kidneys, use a 100 &#181;m cell strainer, two 50 ml tubes, and a magnet catcher.</li>
	<li>Prepare CHAPS lysis buffer: 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 20 mM Tris(hydroxymethyl)-aminomethan (Tris) pH 7.5; 50 mM sodiumchorlide (NaCl); 50 mM sodiumfluoride (NaF); 15 mM Na4P2O7; 0.1 mM Ethylenediaminetetraacetic acid (EDTA) pH 8.0; 2 mM sodiumorthovanadate; and 2 mM adenosinetriphosphat (ATP).</li>
	<li>Cool centrifuges to 4 &#176;C.</li>
</ol>
2. Surgery Under the Microscope
<ol>
	<li>Anesthetize the mouse (e.g., with ketamine/xylazine intraperitoneally, see step 1.11). Perform the toe-pinch test to confirm proper anesthesia.</li>
	<li>Disinfect the ventral side of the mouse with 70% isopropanol.</li>
	<li>Perform a median cut through the skin from the pelvis to the sternum and remove the skin from the abdominal fascia using tweezers. Cut the skin on both sides in the middle of the abdomen with surgical scissors.</li>
	<li>Change surgical tools. Apply a median cut from xiphoid to the bladder through the abdominal muscle layer and divide them into four quadrants using tweezers and surgical scissors. Attach the upper two aside with clamps toward the neck of the mouse with two surgical clamps. 
	NOTE: The abdomen is now opened.</li>
	<li>Put visceral organs aside with a sterile swab and cut the hepatic-phrenic ligament with fine scissors.</li>
	<li>Prepare a ligation (with a silk thread 4-0 to 6-0) around the aorta proximal of the renal arteries on the height of the adrenal gland with two fine tweezers.&#160;Tighten the proximal aortic ligation to abolish blood flow with tweezers.</li>
	<li>Free the distal aorta from fascia, fat, and other tissues and prepare a ligation around the hepatic/mesenteric artery cranial of the renal arteries using fine surgical tweezers.</li>
	<li>Prepare a ligation (with a silk thread 4-0 to 6-0) around the aorta distal of the renal arteries and clamp the vena cava and aorta at the height of the bifurcation.</li>
	<li>Cut a small hole into the aorta distal to the renal arteries so that the hole is half the diameter of the aorta. Put the catheter into the aorta and fix it with the prepared ligature. 
	CAUTION: Avoid bubbles in the perfusion system to prevent air embolism.</li>
	<li>Start perfusion with the ice-cold PBSCM at a flow rate of 2 mL/min.</li>
	<li>Cut a hole into the renal vein at the level of the renal arteries with fine surgical scissors and tighten the ligation around the hepatic and mesenteric arteries. 
	NOTE: Kidneys should turn pale after starting the perfusion.</li>
</ol>
3. In Vivo Biotinylation
<ol>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 0.5 mg/mL biotin for surface labelling, and perfuse kidneys with 5 mL at a flow rate of 2 mL/min. 
	NOTE: Mix the PBSCM solutions (e.g., by turning the 50 mL tubes) gently to avoid forming bubbles within the solution. After aspiration of the solution within the syringe, evacuate any bubbles or air from the syringe. Use an extra cannula (21G) on the syringe to fill the space in the cannula that is connected to the catheter system. Finally, stick the syringe into the cannula with the catheter without bubbles.</li>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 100 mM glycine to quench the glomeruli and perfuse with 5 mL at a flow of 2 mL/min.</li>
	<li>Change the syringe bubble-free to ice-cold PBSCM supplemented with 200 &#956;L magnetic beads/mL and perfuse kidneys at 2 mL/min. 
	NOTE: On the kidney surface, embolization of glomeruli with brown magnetic beads will be visible.</li>
</ol>
4. Isolation of Glomeruli from Two Kidneys
<ol>
	<li>Remove kidneys at the hilum and remove the capsule. Place the kidneys on ice in 15 mL of PBSCM in a 10 cm cell culture dish.&#160;Euthanize the mouse with cervical dislocation under anesthesia after harvesting the kidneys. The surgery and perfusion procedures last approximately 20-22 min.</li>
	<li>Cut kidneys into the smallest pieces possible with a new double-edge blade. Move the tissue into a 2 mL tube with 1 mL of collagenase A (see step 1.6) and digest at 37 &#176;C for 30 min. Before and after digestion, mix gently with a 1000 &#956;L cut pipette.</li>
	<li>Rinse the digested tissue through a 100 &#956;m cell strainer using ice-cold PBSCM. Gently use the cell-scraper to mince the remaining tissue trough the cell strainer and rinse it with ice-cold PBSCM afterwards to a total volume of 50 mL.</li>
	<li>Centrifuge the suspension at 4 &#176;C 500 x g for 5 min. Remove the supernatant and resuspend the pellet with slightly less than 1.5 mL of ice-cold PBSCM while vortexing the pellet. Afterwards, put the glomerular suspension into a new 2 mL tube.</li>
</ol>
5. Washing
<ol>
	<li>Use the magnet catcher to pull the glomeruli to one side. Wait for 1 min before the glomeruli have moved toward the magnet.</li>
	<li>Remove the supernatant with a 1000 &#956;L pipette while the 2 mL tube remains in the magnet catcher. During the first and second washing steps (see step 5.3), 250 &#956;L of supernatant remains in the tube to avoid the loss of glomeruli. Perform this procedure quickly to avoid letting tubular structures sink to the bottom of the tube. 
	NOTE: The washing procedure will last longer otherwise.</li>
	<li>Remove the 2 mL tube from magnet catcher and add 1 mL of PBSCM, pipette up and down (very important), then vortex.</li>
	<li>Put the tube back into the magnet catcher and start over with step 5.2.</li>
	<li>Repeat the washing steps until the purity of glomeruli has reached 90%. For this, examine a representative aliquot of the supernatant under a microscope (40-100X). 
	NOTE: Glomeruli will appear as round structures containing brown magnetic beads. Elongated structures are tubular fragments, and free magnetic beads appear as brown round dots. Cell debris might appear as bulky structures.</li>
	<li>If purity is reached, estimate the count of glomeruli by dissolving the glomeruli in 1 mL of PBSCM. After mixing, take an aliquot of 10 &#956;L and count the glomeruli under the microscope. Calculate the number of glomeruli with the following equation: final number of glomeruli = number of glomeruli in 10 &#956;L aliquot x 100.</li>
	<li>Successful isolation of glomeruli leads to a range of 10,000-40,000 glomeruli.</li>
</ol>
6. Protein Extraction and Immunoprecipitation (IP)
<ol>
	<li>Collect glomeruli by centrifugation at 4 &#176;C at 6800 x g for 5 min. Remove the supernatant while using a magnet catcher and resuspend the pellet in ice-cold lysis buffer (e.g., 30,000 glomeruli in 300 &#956;L; CHAPS, see step 1.11). Homogenize the samples with a tissue homogenizer at the highest speed for 30 s and lyse them on ice for 30 min.</li>
	<li>Remove insoluble material by centrifugation at 15,000 x g for 30 min for 4 &#176;C. Pipette the supernatant which includes the cell lysate into a new 1.5 mL tube. Discard the pellet.</li>
	<li>Measure the protein concentration of the supernatant by using a bicinchoninic (BCA)-based method following the manufacturer&#39;s instructions. A successful lysis of 30,000 glomeruli yields to a glomerular protein amount of 700-1,000 &#181;g/mL. Adjust to equal protein amounts with lysis buffer.</li>
	<li>Take an aliquot of 10% of the total volume for glomerular lysate and add 2x Laemmli + dithiothreithol (DTT) before incubation for 5 min at 95 &#176;C.</li>
	<li>For IP, incubate the rest of the lysate with streptavidin agarose beads overnight at 4 &#176;C on an overhead shaker.</li>
	<li>Centrifuge agarose beads at 1000 x g for 3 min at 4 &#176;C, remove the supernatant, and add 800 &#956;L of lysis buffer to wash the beads for unspecific protein binding.</li>
	<li>Repeat the wash 3 times, and remove the supernatant completely.</li>
	<li>Add 30 &#956;L of 2x Laemmli + DTT and incubate at 95 &#176;C for 5 min.</li>
	<li>Load the lysate and IP probes on a 10% SDS gel and run the SDS gel at 70 V for 30 min, then at 20 mA per gel for 1.5 h. Afterwards, blot the gel for 2 h at 200 mA on a nitrocellulose membrane.</li>
	<li>Block the nitrocellulose membrane overnight at 4 &#176;C with 5% bovine serum albumin (BSA) in washing buffer.</li>
	<li>Incubate the membrane with the antibody of interest overnight at 4 &#176;C. Wash with washing buffer 3 times for 5 min on a shaker and incubate the membrane with the HRP-tagged secondary antibody for 1 h at room temperature. Repeat the washing step.</li>
	<li>Visualize the lysate and immunoprecipitation probes on the membrane by using a super-resolution chemiluminescent agent with a CCD camera.</li>
</ol>

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The authors have nothing to disclose.

The presented method enables successful isolation of glomeruli to investigate glomerular RNA or protein. In addition, primary glomerular cell cultures can be performed from the isolated glomeruli. If biotin is applied before glomerular isolation, labeling of glomerular cell surface proteins can be performed. With this method, in vivo glomerular cell surface protein trafficking can be studied, and semi-quantitation of protein abundance is possible. The most critical steps for successfully testing glomerular cell ...

Proteinuria is a hallmark of glomerular injury and usually accompanies disruption of the glomerular filter1. The glomerular filter is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes. The delicate molecular structure of the glomerular filter is highly dynamic and subject to cell surface protein trafficking in both healthy and diseased kidneys2,3,4,5,6. Endocytosis of cell surface proteins has been shown to be essential for the survival of podocytes7. Nephrin and podocalyxin are transmembrane proteins expressed on podocytes. Nephrin is the backbone of the glomerular slit diaphragm, while podocalyxin is a sialoglycoprotein coating the secondary foot processes of podocytes8,9,10. Endocytic trafficking has previously been shown for nephrin and podocalyxin3,11,12,13,14.
To the best of our knowledge, endocytosis of cell surface proteins has not yet been described in glomerular endothelial cells in the literature. However, endothelial cells in general express all necessary proteins for the different types of endocytosis (i.e., clathrin-dependent, raft-dependent endocytosis)15,16. Therefore, endothelial cell surface trafficking may be studied with this method using, for example, vascular endothelial (VE)-cadherin and intracellular adhesion molecule (ICAM-2) as a cell surface marker protein for glomerular endothelial cells17.
Unfortunately, there is no accurate in vitro model for the delicate three-layered glomerular filter in which cell surface protein trafficking can be studied. The goal of this method is thus to study glomerular protein trafficking in vivo. In addition, this protocol contains information on how to isolate glomeruli, enabling further analysis of glomerular RNA, proteins, or cells. Similar glomerular isolation techniques have been described by different groups18,19.
Previously, we and others have used ex vivo labeling of glomerular cell surface proteins by biotinylation2,3,4,20,21. However, in this ex vivo method, isolated glomeruli were exposed to mechanical stress, which may influence endocytic trafficking. Alternatively, immunofluorescence labeling of glomerular cell surface proteins has extensively been used in the literature2,20,22. With this method, however, only a small number of proteins can be analyzed within one slide, and quantitation of immunofluorescence images is often difficult.
This novel in vivo method offers a reliable tool to study glomerular cell surface protein abundance and trafficking accurately in healthy and diseased kidneys, and it can be used as an addition to immunofluorescence tests.

<table border="0" cellpadding="0" cellspacing="0" style="width:671px;" width="669">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Motic SMZ168 BL</td>
			<td style="width:77px;">Motic</td>
			<td style="width:101px;">SMZ168BL</td>
			<td style="width:235px;">microscope for mouse surgery</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">KL1500LCD</td>
			<td style="width:77px;">Pulch and Lorenz microscopy</td>
			<td style="width:101px;">150500</td>
			<td>light for mouse surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td>PZN:01320422</td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Microfederschere</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">FD100R</td>
			<td>fine scissors, for cut into the aorta</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Durotip Feine Scheren</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BC210R</td>
			<td>for abdominal cut</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Anatomische Pinzette</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BD215R</td>
			<td>for surgery until the abdomen is opened</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Pr&#228;parierklemme</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">BJ008R</td>
			<td>for surgery&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Seraflex</td>
			<td style="width:77px;">Serag Wiessner</td>
			<td style="width:101px;">IC108000</td>
			<td>silk thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ketamine 10%</td>
			<td style="width:77px;">Medistar</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/100</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/200</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Harvard apparatus 11 Plus</td>
			<td style="width:77px;">Harvard Apparatus</td>
			<td style="width:101px;">70-2209</td>
			<td>syringe pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EZ-link Sulfo-NHC-LC-Biotin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21335</td>
			<td>biotin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Dynabeads Untouched Mouse T-cells</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">11413D</td>
			<td>to embolize glomeruli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Collagenase A</td>
			<td style="width:77px;">Roche</td>
			<td style="width:101px;">10103578001</td>
			<td>to digest kidney tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DynaMag-2</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">123.21D</td>
			<td>Magnet catcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">100&#181;m cell stainer</td>
			<td style="width:77px;">Greiner-bio</td>
			<td style="width:101px;">542000</td>
			<td>for glomerular isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Axiovert 40 CFL</td>
			<td style="width:77px;">Zeiss</td>
			<td style="width:101px;">non available</td>
			<td>to confirm glomerular purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">TissueRuptor</td>
			<td style="width:77px;">Quiagen</td>
			<td style="width:101px;">9002755</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">CHAPS</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">C3023</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Tris-HCL</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">T5941</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaCl</td>
			<td style="width:77px;">VWR chemicals</td>
			<td style="width:101px;">27810295</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaF</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">201154</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EDTA</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">E5134</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">ATP</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">34369-07-8</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td>Follow the manufacturer&#39;s instructions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">nephrin antibody</td>
			<td style="width:77px;">Progen</td>
			<td style="width:101px;">GP-N2</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Polyclonal goat anti-podocalyxin antibody</td>
			<td style="width:77px;">R&#38;D Systems</td>
			<td style="width:101px;">AF15556-SP</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Streptavidin Agarose Resin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">20347</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Protein A sepharose CL-4B</td>
			<td style="width:77px;">GE Healthcare</td>
			<td style="width:101px;">17096303</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">polyclonal rabbit anti-p57 antibody</td>
			<td style="width:77px;">SCBT</td>
			<td style="width:101px;">sc-8298</td>
			<td>for Immunohistochemistry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">mouse monoclonal anti-beta actin antibody, clone AC-74</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2228</td>
			<td>Western blot loading control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">rabbit anti-p44/42</td>
			<td style="width:77px;">cell signalling</td>
			<td style="width:101px;">4695</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce High sensitivity streptavidin-HRP</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21130</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse ICAM-2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF774</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse anti-VE-cadherin</td>
			<td>R&#38;D Systems</td>
			<td>AF1002</td>
			<td>for westernblot</td>
		</tr>
	</tbody>
</table>

isolation of glomeruli and in vivo labeling of glomerular cell surface proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

To isolate glomeruli accurately, it is necessary to perfuse murine kidneys with PBSCM first. Perfusion with PBSCM turns kidneys pale (Figure 1A). Embolization of glomeruli with magnetic beads will be visible as brown dots on the kidney surface (Figure 1B). Isolation of glomeruli with the magnet catcher may show contamination with renal tubuli (Figure 1C</stro...

Watch this Scientific Journal Video about Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins at JoVE.com

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

isolation-glomeruli-vivo-labeling-glomerular-cell-surface

Research

JoVE Journal

Biochemistry

9.0K Views.  Heinrich-Heine-University. Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Video: Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Summary

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Chapters in this video

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography