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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
Protein–ligand or protein–protein interaction regulates various biological processes. Because these interactions are often mediated by noncovalent bond, the detection of the complexes should be performed under the conditions where the complexes remain intact. Gel filtration chromatography has been historically used for the analysis of such complexes. However, this method has some drawbacks, such as a low throughput, dilution effects, and requiring large volumes of buffer and relatively large amounts of specimens. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an alternative method for analyzing macromolecular complexes. It is relatively high throughput and ideal for the downstream experiments, such as multi-dimensional electrophoresis, isolation of the complexes, and western blot. In BN-PAGE, the samples are mixed with a negatively charged dye Coomassie Brilliant blue G-250 (not R-250) for the crude ones or Ponceau S for the purified complex, which binds to proteins and allows for separating the complexes according to their molecular sizes during electrophoresis. BN-PAGE has been successfully applied to resolve membrane protein complexes, such as oligosaccharyltransferases and the protein-conducting channels (1, 2), which are required for the biosynthesis of N-glycosylated proteins in the endoplasmic reticulum of eukaryotic cells (3).
Protocol
Reagents required for BN-PAGE are commercially available from Invitrogen (Bis-Tris/tricine buffer system). To prepare the reagents in your lab, please see below (Immidazole/tricine buffer system). All the procedures are based on the previous report (4).
Materials
- 1.
1 M imidazole/HCl, pH 7.0; store at 4°C.
- 2.
1 M imidazole (non-pH-adjusted stock); store at 4°C.
- 3.
10% (w/v) digitonin (Merck, Darmstadt, Germany) (see Note 1).
- 4.
1 M tricine, store at 4°C
- 5.
2 M 6-aminohexanoic acid; store at 4°C
- 6.
Acrylamide-bisacrylamide mix (AB mix; 49.5% T and 3% C) (see Note 2), store at 4°C.
- 7.
3× Gel buffer (75 mM of imidazole/HCl, pH 7.0 and 1.5 M 6-aminohexanoic acid); store at 4°C
- 8.
10% (w/v) ammonium persulfate (APS); store at 4°C
- 9.
N,N,N',N'-tetramethylethylenediamine (TEMED); store at 4°C
- 10.
5% (w/v) Coomassie Brilliant blue G-250 in 500 mM of 6-aminohexanoic acid; store at room temperature.
- 11.
Ponceau S/glycerol stock (0.1% Ponceau S and 50% glycerol, w/v); store at 4°C.
- 12.
50% (w/v) glycerol; store at 4°C.
- 13.
Cathode buffer + Dye (50 mM of tricine, 7.5 mM of imidazole [non-pH adjusted], and 0.02% Coomasie blue G-250); prepare when used and chill at 4°C.
- 14.
Cathode buffer + Dye/10 (50 mM of tricine, 7.5 mM of imidazole [non-pH adjusted], and 0.002% Coomassie blue G-250); prepare when used and chill at 4°C
- 15.
Anode buffer (25 mM of imidazole, pH 7.0); prepare when used and chill at 4°C
- 16.
High molecular weight native marker kit (Cytiva, Amersham Place, UK)
Instruments
- 1.
Conventional apparatuses for polyacrylamide gel electrophoresis (see Note 3)
- 2.
Gradient mixer (e.g., Hoefer™ SG Series Gradient Makers, Hoefer™ SG50).
- 3.
Peristaltic pump (e.g., ATTO Perista Pump, 7–700 mL/h).
Methods
- 1.
Preparation of native polyacrylamide gel
- a.
Mix the reagents on ice (Critical Point), listed in Table 1, except for APS and TEMED.
- b.
Set up the gradient mixer and peristaltic pump (Figure 1A). Put a small stir bar into chamber A.
- c.
Add APS and TEMED to the 4% separating gel solution and pour 3.75 mL of the solution into chamber A.
- d.
Fill the connecting tube with the solution.
- e.
Slant the gradient mixer to lean the solution into chamber B.
- f.
Close the cock.
- g.
Add APS and TEMED to the 13% separating gel solution and pour 3.50 mL of the solution into chamber A.
- h.
Start stirring and pumping.
- i.
Open the cock on the connecting tube.
- j.
Open the cock between chamber A and the peristaltic pump.
- k.
Pour the gradient solution into the gel plates.
- l.
When the solutions are poured into the gel plates, overlay a small volume of water on top of the gradient solution.
- m.
When polymerized, pour the stacking solution that has been mixed with APS and TEMED.
- n.
Insert a comb and wait until polymerized.
- 2.
Sample preparation for BN-PAGE
- a.
Prepare samples of interest in 10–50 mM of buffer, pH 7.0–7.5, containing NaCl less than 50 mM. Add nonionic detergents (e.g., 1% digitonin at final concentration), if necessary.
- b.
Centrifuge at 15,000 ×g for 5 min and recover the supernatant.
- c.
If the sample is purified proteins, add one tenth of the volume of Ponceau S/glycerol stock to the sample just before loading onto the gel. If the sample is crude, add one fifth of the volume of 50% glycerol and one twelfth of the volume of 5% Coomassie G250 to the sample just before loading onto the gel. Add nonionic detergents (e.g., digitonin) at 1%, if necessary.
- 3.
BN-PAGE
- a.
Set the BN gel to the PAGE apparatus.
- b.
Pour the cathode buffer + Dye into the inner chamber.
- c.
Pour the anode buffer into the outer chamber.
- d.
Load the sample into the well. If it is difficult to see the well, use a backlight.
- e.
Run the gel in a refrigerator or in a cold room with a constant voltage at 100 V.
- f.
When the samples have completely entered the gel, change the setting to a constant current at 15 mA until the dye front reaches approximately one third from the top of the gel.
- g.
Aspirate the cathode buffer + Dye completely and pour the cathode buffer + Dye/10.
- h.
Stop the electrophoresis when the dye front reaches the bottom of the gel.
- 4.
Staining
- a.
If the amounts of the sample are sufficient, you will see the protein bands immediately after the electrophoresis.
- b.
If you need to stain the gel with silver, destain the gels overnight with methanol/acetate/water (5:1:4, v/v/v) and then perform the silver staining (Figure 1B).
- 5.
Western blot
- a.
Perform the regular electroblotting with polyvinylidene fluoride membrane. (Do NOT use nitrocellulose membrane.)
- b.
After the electroblotting, wash the membrane three times with water, and destain it with methanol.
- c.
After the destaining, wash the membrane three times with phosphate-buffered saline that contains 0.05% Tween 20 and proceed to western blot. Depending on antibodies, they may not bind to the antigens that retain their native conformation or are embedded in the structural part of the proteins. In these cases, the antibodies may recognize the antigens after the membrane was denatured for 30 min at 50°C in a buffer containing 62.5 mM of Tris-HCl, pH 6.8, 2% SDS, and 0.8% β-mercaptoethanol.
Table 1:
Compositions of the stacking gel and the gradient separating gel for a minigel.
Notes
- 1.
Aliquot in a small volume and store at −80°C.
- 2.
“% T” indicates the total concentration of acrylamide and bisacryamide, and “% C” indicates the percentage of bisacrylamide. Mix 48 g of acrylamide and 1.5 g of bisacrylamide in 100 mL of water.
- 3.
Detergents must be completely washed out.
References
- 1.
- Harada Y, Li H, Li H, Lennarz WJ. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site. Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):6945–49. [PMC free article: PMC2678446] [PubMed: 19365066] [CrossRef]
- 2.
- Harada Y, Li H, Wall JS, Li H, Lennarz WJ. Structural Studies and the Assembly of the Heptameric Post-translational Translocon Complex. J Biol Chem. 2011 Jan 28;286(4):2956–65. [PMC free article: PMC3024790] [PubMed: 20826819] [CrossRef]
- 3.
- Harada Y, Ohkawa Y, Kizuka Y, Taniguchi N. Oligosaccharyltransferase: A gatekeeper of health and tumor progression. Int J Mol Sci. 2019 Dec 2;20(23):6074. [PMC free article: PMC6929149] [PubMed: 31810196] [CrossRef]
- 4.
- Wittig I, Braun HP, Schägger H. Blue native PAGE. Nat Protoc. 2006 Jun 27;1(1):418–28. [PubMed: 17406264] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
- Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.[Anal Biochem. 2014]Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.Raghupathy V, Oommen A, Ramachandran A. Anal Biochem. 2014 Jun 15; 455:1-2. Epub 2014 Mar 22.
- Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer.[J Biol Chem. 1985]Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer.Tal M, Silberstein A, Nusser E. J Biol Chem. 1985 Aug 25; 260(18):9976-80.
- Gel-electrophoresis based method for biomolecular interaction.[Methods Cell Biol. 2022]Gel-electrophoresis based method for biomolecular interaction.Arakawa T, Nakagawa M, Tomioka Y, Sakuma C, Li C, Sato T, Sato R, Shibata T, Kurosawa Y, Akuta T. Methods Cell Biol. 2022; 169:67-95. Epub 2022 Feb 2.
- Analysis of the enzymatic degradation of lysozyme fibrils using a combination method of non-denaturing gel electrophoresis and double staining with Coomassie Brilliant Blue G-250 and R-250 dyes.[Anal Sci. 2023]Analysis of the enzymatic degradation of lysozyme fibrils using a combination method of non-denaturing gel electrophoresis and double staining with Coomassie Brilliant Blue G-250 and R-250 dyes.Nagata K, Ashikaga R, Mori W, Zako T, Shimazaki Y. Anal Sci. 2023 Mar; 39(3):267-274. Epub 2022 Nov 30.
- Review [Protein determination by binding with the dye Coomassie brilliant blue G-250].[Biokhimiia. 1994]Review [Protein determination by binding with the dye Coomassie brilliant blue G-250].Gasparov VS, Degtiar' VG. Biokhimiia. 1994 Jun; 59(6):763-77.
- Blue native polyacrylamide gel electrophoresis - Glycoscience Protocols (GlycoPO...Blue native polyacrylamide gel electrophoresis - Glycoscience Protocols (GlycoPODv2)
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