Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Summary

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Abstract

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

Introduction

Normal cellular function is dependent on protein-protein interactions^1,2. As a result, human diseases are often marked by perturbations in the assembly and behavior of various protein complexes³. The ability to characterize such interactions is therefore critical. Current means of detecting these interactions require purification of target proteins, often followed by pull-down of their interacting partners. Conventional purification is accomplished by differential centrifugation, precipitation, and/or chromatography⁴. These methods are time-consuming, must be altered for each target protein, and often result in low yields. Modern purification methods involve the fusion of peptide tags to target proteins, followed by immunoprecipitation or extraction on columns loaded with beads bound to a capture molecule^5,6. While this is extensible to many proteins, it requires sequence modification of the target, potentially resulting in constructs that differ in affinity for their usual binding partners. The delicate nature of some protein-protein interactions means this method may not be applicable to many scenarios. Additionally, the pull-down assays used to map protein-protein interactions may not capture an accurate cellular picture, due to restricted degrees of freedom and non-native levels of the bait protein.

Ideally, protein complexes could be detected in their native states, without the need for purification or pull-down. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was developed as a less-denaturing alternative to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and allows for the separation of some proteins and complexes from biological samples⁷. However, proteins in BN-PAGE separate based on a large number of variables, including size, charge, three-dimensional structure, and association with other molecules. The interactions of these factors often result in co-separation of proteins, aggregate formation, and poor protein band resolution. Two-dimensional native-polyacrylamide gel electrophoresis resolves some, but not all, of these problems⁸.

To circumvent the complications associated with native separation, some authors use amine-reactive cross-linking reagents, such as dithiobis(succinimidyl propionate) (DSP), to capture protein complexes in tissue lysates⁴. These treated lysates can then be denatured and separated by SDS-PAGE, while preserving the native size and makeup of protein complexes. However, since cross-linking reagents react based on proximity of one molecule to another, and proteins in tissue lysates have many degrees of freedom and can stochastically interact, nonspecific background cross-linking can be high, especially in concentrated samples. This can lead to difficult-to-interpret results.

Here, we demonstrate the use of a hybrid BN-PAGE/SDS-PAGE method, termed multimer-polyacrylamide gel electrophoresis (multimer-PAGE), to separate and detect protein assemblies in complex mixtures. Initially, cell lysate is suspended in polyacrylamide gel via BN-PAGE. The lysate-containing gel is then reacted with the cross-linking reagent DSP. Pseudo-immobilized and slightly separated on the gel, proteins are much less likely to react nonspecifically, meaning background cross-linker reactivity is reduced. After cross-linking, the gel bands are excised and separated via SDS-PAGE. The resultant gel can then be analyzed by any means typically associated with polyacrylamide gel electrophoresis. This method allows for the separation and detection of native protein complexes in unmodified tissue lysate, without the need for additional purification or pull-down.

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Protocol

1. Tissue Preparation

1. Prepare 10 mL of 4x BN-PAGE sample buffer (200 mM Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), 200 mM NaCl, 40% w/v glycerol, 0.004% Ponceau S, pH 7.2).
   NOTE: This stock solution may be made in advance and stored at 4 °C.
2. Dilute 250 µL of 4x sample buffer in 750 µL dH₂O containing 1x commercial protease inhibitor cocktail. Vortex and chill on ice.
3. Homogenize 20 mg of target tissue in the 1 mL 1x ice-cold BN-PAGE sample buffer with 30 strokes of a clean dounce homogenizer.
   NOTE: For this demonstration experiment, the target tissue is whole rat brain tissue. After homogenization, samples may be treated with mild detergent such as 2% digitonin to solubilize and permit electrophoresis of membrane proteins.
4. Transfer the homogenate to a 1.5 mL microcentrifuge tube, and centrifuge at 14,000 x g for 30 min to pellet insoluble cellular contents. After centrifugation, decant the supernatant into a clean tube on ice.
5. Follow manufacturer instructions to measure supernatant protein concentration using bicinchoninic acid (BCA) or similar protein quantitation assay.
6. If the homogenate sample(s) contain detergent, add a quantity of 5% Coomassie Blue G-250 in aqueous solution sufficient to bring the homogenate solution to 0.25% Coomassie.

2. BN-PAGE

1. Dilute 25 mL 20x blue native (BN) electrophoresis buffer stock (1.0 M Bis-Tris, 1.0 M tricine, pH 6.8) into 475 mL dH₂O containing 0.002% Coomassie Blue to make 500 mL of 1x running buffer.
   1. If samples contain detergent, instead make 250 mL of 1x running buffer containing 0.02% Coomassie and another 250 mL containing none.
2. Chill buffer(s) to 4 °C.
3. Clean and assemble a polyacrylamide gel pouring cassette according to manufacturer instructions.
4. Pour BN polyacrylamide gel (3% T stacking layer, 6% T resolving layer) according to a standard recipe⁷, and place the well comb.
5. After the gels have polymerized, rinse the cassette with dH₂O and assemble the electrophoresis apparatus according to manufacturer instructions.
6. Remove the well comb and fill the wells with 1x BN-PAGE running buffer. Avoid filling the entire inner chamber with buffer until samples are loaded.
   1. If samples contain detergent, fill the wells with the buffer containing 0.02% Coomassie Blue.
      NOTE: Perform steps 2.7-2.9 at 4 °C.
7. Using gel-loading tips, pipette a volume of homogenate containing 20 µg of protein into the desired wells. Pipette an equivalent volume of 1x BN-PAGE sample buffer into any unused wells.
   NOTE: Use the protein concentration determined from the BCA assay to calculate the appropriate volume of homogenate to add to the wells.
8. After samples are loaded, fill the inner chamber with 1x BN-PAGE running buffer. Be sure the gel is entirely submerged in buffer. Next, fill the outer chamber with 1x running buffer to the level indicated by the manufacturer.
   1. If samples contain detergent, fill the inner chamber with the 1x buffer containing 0.02% Coomassie Blue, and the outer chamber with dye-free 1x running buffer.
9. Connect the electrodes to the power supply, and electrophorese the proteins in the gel at 150 V until the dye band progresses ~2 cm into resolving layer. Stop and disconnect the power supply.

3. Cross-linking

NOTE: Perform Steps 3.1-3.6 at 4 °C.

1. Disassemble the electrophoresis apparatus, and separate the glass panes of the gel cassette. Remove and discard the stacking layer.
2. Carefully cut the gel just below the bottom edge of the dye front. Take care to make this cut as smooth and straight as possible, and then discard the unused piece of gel. This will leave a ~2 cm wide, horizontal strip of polyacrylamide gel, which will contain all the protein in the homogenate sample.
3. Trim away any unused portions along the edges of the gel strip.
4. Carefully place the strip into 10 mL phosphate buffered saline (PBS) in a small container, and gently mix by nutation for 30 min to equilibrate.
5. After equilibration, discard and replace the PBS with another 10 mL. Pipette 500 µL of 25 mM DSP dissolved in dimethyl sulfoxide into the PBS, and continue mixing as above (step 3.4) for 30 min.
6. Pour off the DSP solution. Add 10 mL of 0.375 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.8, containing 2% sodium dodecyl sulfate (SDS) to quench the unreacted DSP. Continue nutation for 15 min.

4. SDS-PAGE

1. While gel strip is quenching, prepare SDS-PAGE gel solutions according to standard methods⁹. Do not add polymerization reagents.
2. After quenching, return the ΒΝ gel strip to room temperature, and cast the strip into a new gel cassette.
   1. To do this, carefully pick up the gel and place it onto a clean gel cassette spacer plate.
   2. Orient the strip so the bottom of the dye front is nearest to the top of new cassette (i.e., the side that travelled furthest during BN-PAGE should be closest to the top of the new cassette; the gel strip should be flipped from its prior orientation). See Figure 3.
   3. Place the strip such that its top edge lies even with where the top edge of the cover plate will be (i.e., it will be at the top of the new gel). Make sure the dye front is parallel to the horizontal edges of the glass plate.
   4. Push one side of the excised strip against one of the spacer walls, leaving room on the other side for gel to be poured and a protein standard or ladder to be loaded.
   5. If the bottom edge of the gel strip contains any jagged or uneven areas, carefully cut them away. If present, they will trap bubbles during gel pouring.
   6. Once the gel strip is positioned correctly, lay the cover plate over the spacer plate. Apply gentle pressure to push out any trapped air bubbles.
   7. Continue to assemble the gel-pouring apparatus according to manufacturer instructions.
3. Add polymerization reagents to the resolving gel buffer, and pour it into the prepared gel cassette, using a serological pipette. Fill the gel cassette to ~2 cm below the excised BN-PAGE gel strip, to leave room for the stacking layer.
4. Add 100 µL butanol over the top of the poured gel, and allow 30 min for polymerization of the resolving layer. Pour off the butanol.
5. Add polymerization reagents to the stacking gel solution. Using a serological pipette, pour the stacking layer to fill all remaining empty space in the gel cassette.
   1. Tilt the gel cassette as the stacking layer is poured so it fills the space below the gel strip, and air bubbles are not trapped.
   2. As the stacking gel buffer fills the empty space below the gel strip, gradually return the gel cassette to level footing.
   3. Continue to fill the empty space next to the excised gel strip with the stacking gel buffer, until it nearly overflows.
6. Allow the stacking layer to polymerize for 30 min.
   NOTE: Make 10x SDS-PAGE running buffer (250 mM tris(hydroxymethyl)aminomethane (tris), 1.9 M glycine, 1% SDS) while the gel is polymerizing. This can also be made beforehand and stored at room temperature.
7. Dilute 50 mL 10X SDS-PAGE running buffer stock into 450 mL dH₂O to make 1x working buffer.
8. After the stacking layer has polymerized, remove the gel cassette from the pouring apparatus, rinse with dH₂O, and assemble the electrophoresis apparatus according to manufacturer instructions.
9. Fill the inner chamber completely with 1x SDS-PAGE running buffer, then fill the outer chamber to the level indicated by the manufacturer.
10. Load the space next to the excised gel strip with a molecular weight ladder or appropriate protein standard.
11. Attach the electrodes to the power supply, and electrophorese the samples at 120 V. When the Coomassie dye runs off the gel, stop and disconnect the power supply.
12. Analyze the gel using standard methods of electroblotting and protein detection by antibody binding¹⁰.

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Results

In this demonstration experiment, multimer-PAGE was performed on whole rat brain lysate. The resultant separated proteins were blotted onto polyvinylidene diflouride (PVDF) membranes, and then probed with antibodies against proteins that are known to form complexes. Figure 1 shows a validation of the protocol by two means. First, we demonstrate that the cross-linked proteins are cleavable by addition of a reducing agent, meaning the observed higher molecular weight specie...

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Discussion

Protein-protein interactions are important for every task living things carry out. Because of this, they are the subject of intense scrutiny and research. Multimer-PAGE is a novel method for capturing, separating, and analyzing a wide range of protein complexes. We have previously demonstrated its applicability to studying oligimerization of the disease-associated protein α-synuclein¹¹. However, it is extensible to many protein complexes, as demonstrated in Figure 2. When com...

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Materials

Name	Company	Catalog Number	Comments
Chemicals
ε-Aminocaproic acid	Sigma	A2504
Acrylamide	Acros Organics	164855000	Toxic.
Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)	BioRad	161-0148	Toxic.
Ammonium persulfate	Sigma	A3678
Anti rabbit IgG-HRP from goat	Santa Cruz Biotechnology	sc-2004
Bicinchoninic acid assay kit	Thermofisher	23225
Bis-tris	Sigma	B9754
Bovine serum albumin	Sigma	A9647
Butanol	Fisher Scientific	A399-1
Chemiluminescence substrate kit	ThermoFisher	24078
Coomassie blue G-250	Sigma-Aldrich	B0770
Digitonin	Sigma	D141	Toxic.
Dimethyl sulfoxide	Fisher Scientific	D128-1
Dithiobis(succinimidylpropionate)	Thermo Scientific	22585
Dry nonfat milk	LabScientific	M0841
Glycerol	Sigma	G9012
Glycine	Fisher Scientific	BP381-5
Halt Protease Inhibitor Cocktail	Thermofisher	78430
Hydrochloric acid	Fisher Scientific	A144SI-212	For titration. Caustic.
Methanol	Fisher Scientific	A412-4	For PVDF membrane activation. Toxic.
Monoclonal anti α-synuclein IgG from rabbit	Santa Cruz Biotechnology	sc-7011-R
N,N,N',N'-tetramethylethylenediamine	Sigma	T9281
N,N'-methylenebisacrylamide	Acros Organics	16479	Toxic.
NP40	Boston Bioproducts	P-872
Polysorbate 20 (tween-20)	Fisher Scientific	BP337-500
Polyvinylidene fluoride transfer membranes	Thermo Scientific	88518
Ponceau S	Sigma	P3504
Potassium chloride	Fisher Scientific	P217-3
Potassium phosphate monobasic	Sigma	P9791
Protease inhibitor cocktail	Thermo Scientific	88265
SDS solution (10% w/v)	BioRad	161-0416
Sodium chloride	Fisher Scientific	BP358-212
Sodium dodecyl sulfate	Sigma	L37771
Sodium phosphate monobasic	Fisher Scientific	BP329-1
Tricine	Sigma	T0377
Tris base	Fisher Scientific	BP152-500
Tris-HCl (0.5 M buffer, pH 6.8)	BioRad	161-0799
Tris-HCl (1.5 M buffer, pH 8.8)	BioRad	161-0798
Name	Company	Catalog Number	Comments
Instruments
GE Imagequant LAS 4000	GE Healthcare	28-9558-10
ImageJ software	NIH
Synergy H1 microplate reader	BioTek
Gel Former + Stand	Biorad
Microfuge 22R centrifuge	Beckman Coulter
2 mL dounce homogenizer
Vortex mixer	Fisher Scientific
Ultrasonic tissue homogenizer	Fisher Scientific	FB120220