Non-Reducing SDS PAGE: A Method to Analyze Disulfide-Linked Multimeric Protein Complexes

Overview

This video describes the technique of separating the protein samples by non-reducing SDS-PAGE to analyze the multimeric protein complexes. This technique retains the multimer subunits of a protein held by the disulfide bonds which can later be analyzed by western blotting.

Protocol

1. Blocking Free Cysteine Residues Using Iodoacetamide

1. Grow U-2 OS cells in a 6 cm² dish to 50%–60% confluency in minimum essential medium with high glucose containing 10% fetal bovine serum and 1% sodium pyruvate at 37 °C in 5% CO₂.
2. Make a fresh stock of 10 mM iodoacetamide just prior to use, then discard any unused reagent.
3. Add 0.1 mM final concentration iodoacetamide directly to the cell culture media. Gently rock the dish at room temperature for 2 min.

2. Harvesting the Cells

1. Aspirate the media from the cells.
2. Wash the cells with 5 mL of cold phosphate-buffered saline (PBS) three times. Aspirate the final PBS wash solution then add 1 mL of cold PBS.
3. Scrape the cells off the bottom of the dish using a cell scraper. Using a 1 mL pipette draw up the PBS and cell suspension and dispense all the liquid into a 1.5 mL microcentrifuge tube.
4. Spin the cells at 7,500 x g for three min at 4 °C. Aspirate the PBS, leaving the cell pellet behind. The cell pellet can be stored at -80 °C until processing

3. Extraction of Protein

1. Prepare 100 µL of a 1x lysis buffer diluted in ddH₂O (See Table of Materials). Add phenylmethylsulfonyl fluoride (PMSF) immediately before use to a 1 mM final concentration.
2. Add 50 µL of the 1x lysis buffer directly to cell pellet and suspend. Incubate this on ice for 5 min.
3. Sonicate for 8 s using the constant pulse mode at 40% (see Table of Materials for sonicator; adjust as necessary for different sonicators), keeping the extract on ice.
4. Spin the extract at 4 °C for 5 min at 16,000 x g. The supernatant is the soluble protein fraction. Bradford analysis can be performed to determine protein concentration if desired.

4. Sample Preparation

1. Take 10 µL of the soluble protein extract and add 10 µL of a 1x Laemmli SDS-sample buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl, pH 6.8). Do not add any reducing reagents.
2. Keep samples on ice if SDS PAGE analysis will be performed that day; for long term storage, -20 °C is appropriate. Right before running the gel, heat the sample for 5 min at 85 °C.

5. In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells

1. Grow U-2 OS cells in a 175 cm² flask to 70%–80% confluency.
2. Perform the formaldehyde crosslinking reaction
   1. In a fume hood, aliquot a 37% formaldehyde solution purchased from commercial sources. Add the formaldehyde fixative directly to the medium to a final concentration of 1% and incubate at room temperature with gentle agitation for 15 min.
   2. To quench the reaction, add 1.25 M glycine to a final concentration of 0.125 M and incubate at room temperature with gentle agitation on a rocker for 5 min.
3. Wash the cells with 5 mL of cold PBS three times. Aspirate the final PBS wash solution and add 10 mL of PBS. Scrape the cells off the bottom of the flask using a cell scraper.
4. Using a 10 mL pipette, draw up the PBS and cell suspension and dispense the entire liquid into a 15 mL conical centrifuge tube. Spin the cells at 500 x g for 2 min at 4 °C. Aspirate the PBS, leaving the cell pellet behind.

6. Fractionation of Nuclei

1. Prepare 10 mL of homogenization buffer: 0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, and 0.5% BSA at pH 7.4. Add PMSF immediately before use to a 1 mM final concentration and 3mL of nuclei suspension buffer (0.1% Triton X-100 in PBS).
2. Add 5 mL homogenization buffer directly to the cell pellet and suspend completely. Centrifuge the suspension at 500 x g for 2 min at 4 °C then discard the supernatant.
3. Suspend the pellet in 5 mL of homogenization buffer. Use a tight-fitting glass-Teflon homogenizer, homogenize thecells at 10 strokes/500 rpm.
4. Centrifuge the suspension at 1,500 x g for 10 min at 4 °C, then discard the supernatant.
   NOTE: Use the supernatant for isolation of mitochondria by centrifuging at 10,000 x g for 10 min.
5. Suspend the pellet in 1 mL nuclei suspension buffer and incubate on ice for 10 min.
6. Centrifuge at 600 x g for 10 min. Discard the supernatant.
7. Suspend the pellet in 1 mL nuclei suspension buffer and centrifuge again. Discard the supernatant. The final pellet will be isolated nuclei.

7. Extraction of Protein

1. Repeat section 4, except adding 25 µL of the 1x lysis buffer directly to the cell pellet then suspending.

8. Sample Preparation

1. Prepare two samples for SDS-PAGE by taking 10 µL each of the soluble protein extract and add 10 µL of 2x Laemmli SDS-sample buffer and 1 µL of 2-Mercaptoethanol (BME).
2. Heat one sample for 5 min at 37 °C and the second sample for 15 min at 98 °C to reverse the formaldehyde cross link.

9. SDS-PAGE Analysis

1. Prepare 1 L of 1x Tris-glycine running buffer (25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS).
2. Set up the SDS-PAGE running apparatus.
   NOTE: This protocol uses a 16% precast TGX SDS-Page. Of note, any percentage gel can be used.
   1. Per the manufacturer protocol, open the package the gel is stored in and remove the cassette.
   2. Remove the comb that is lining the wells and the tape from the bottom of the cassette.
   3. Place the gel into the running apparatus.
   4. Fill the chamber with the 1x running buffer until the wells are submerged in liquid. Using a plastic pipette, rinse out the wells with the running buffer.
3. Load the samples on the gel along with 10 µL prestained standard marker.
4. Run the gel at 200 V until the dye front is approximately 1 cm from the bottom of the gel.

Materials

Name	Company	Catalog Number	Comments
16% precast TGX gels	ThermoFisher	Xp00160
Bromophenol Blue
BSA	Cell signaling	99985
Cell lysis buffer	Cell signaling	9803
Centrifuge	Eppendorf	5415D
DMEM	Gibco	11330-032
EDTA	Sigma Aldrich	M101
Electrophoresis apparatus	Invitrogen	A25977
Fetal bovine serum	Gibco	1932693
Formaldehyde	ThermoFisher	28908
Glass-teflon homogenizer
Glycerol	Sigma Aldrich	65516
Glycine	RPI	636050
Hepes	Sigma Aldrich	H0527
Iodoacetamide	ThermoFisher	90034
PBS	Sigma Aldrich	P3813
Power supply	Biorad	200120
Prestained marker	ThermoFisher	26619
SDS	Biorad	161-0302
Sonicator	Branson	450
Tris Base	RPI	T60040
Tris Buffered Saline	Sigma Aldrich	SRE0031	with Tween 20, pH 7.5
Tris-Glycine running buffer	VWR	J61006
Triton X-100	Sigma Aldrich	T8787
U-2 OS	ATCC	HTB-96