Nov 13, 2019
SDS PAGE & Western Blot for Membrane proteins
- 1Department of Chemistry - Ångström, Uppsala
- CyanoWorld
Protocol Citation: Dennis DD Dienst 2019. SDS PAGE & Western Blot for Membrane proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.mhtc36n
Manuscript citation:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10020688.pdf
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026389.pdf
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 09, 2018
Last Modified: November 13, 2019
Protocol Integer ID: 9491
Materials
MATERIALS
2 M Tris-Cl, pH 6.8
DTT (1,4-Dithiothreitol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10708984001
Glycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
20% SDS
Bromophenol blue
PageRuler Prestained Protein Ladder, 10 to 180 kDaThermo Fisher Scientific
Soy Milk (Alpro Soya original)
10 x TGS bufferBio-Rad LaboratoriesCatalog #1610732
Prepare 4 x loading dye
Prepare 4 x loading dye
For 10 mL: final conc.
617 mg DTT 400 mM
1,25 mL 2M Tris-Cl (pH 6.8) 250 mM
4 mL Glycerol 40% (w/v)
4 mL 20% SDS 8% (w/v)
ad H2O to 10 mL
Safety information
Work under the fume hood and wear gloves!
DTT (Dithiothreitol) is flamable and harmful if swallowed or inhaled. Maybe harmful if absorbed through the skin. Causes irritation to skin, eyes and respiratory tract.
SDS (sodium dodecyl sulfate) causes eye and skin irritation and may cause respiratory tract irritation.
Prepare TBS-T buffer
Prepare TBS-T buffer
Stock solutions:
1) 1 M Tris (pH 7.5) - for 1 L: 121,14 g Tris, titrate with HCl
2) 5 M NaCl - for 500 mL: 146,1 g NaCl
For 1 L TBS-T:
100 mL 1 M Tris (pH 7.5)
30 mL 5 M NaCl
1 mL Tween 20
Safety information
When using concentrated HCl (37%), work in the hood! HCl 'smoke' attacks your lungs when inhaled!
Sample denaturation
Sample denaturation
- adjust protein samples to equal concentrations by adding PBS
- add 1 Vol. loading dye (LD) to 3 Vol. sample:
e.g.:
2.5 µL LD + 7.5 µL sample
4 µL LD + 12 µL sample
5 µL LD + 15 µL sample
- incubate 30 min at 50 °C
Note
Do not boil, in order to prevent aggregation !
50 °C
00:30:00
Gel loading
Gel loading
- spin down denatured samples for 1 min at 12000 g
- assemble 'Mini-Protean' chambers and add a mock plate if required
- fill cathode reservoir and chamber (until marker) with 1 x TGS buffer (Biorad)
- remove comb and bottom tape from 'Mini Protean TGX precast gel'
- carefully load sample supernatants into wells using Hamilton syringe or pipette
- load 4 µL 'PageRuler prestained Protein ladder' into one well
Safety information
Work under the fume hood and wear vinyl/PVC gloves ! (cf. step 1)
Acrylamide (non-polymerized) is a neurotoxin which can be absorbed orally, dermally or by inhalation. Signs of exposure include tingling in the extremities and/or loss of coordination.
Electrophoresis
Electrophoresis
- run gel at constant current of 20-40 mA
Western Transfer
Western Transfer
- assemble blotting sandwich according to the Trans-Blot Turbo Transfer System- Quick Sart Guide (link below)
- take care of removing all bubbles (!)
- close and lock cassette lid
- insert cassette into the instrument
Transfer conditions:
Program 'Standard SD' - 30 min (up to 1.0 A; 25 V constant)
Staining
Staining
Blocking
Blocking
- Incubate 15 min with soy milk at room temperature
cf.: Analytical Biochemistry 426 (2012) 22–23 (link below)
Washing/ Equilibration
Washing/ Equilibration
- incubate in TBS-T for 3 x 15 min at room temperature
Primary antibody
Primary antibody
for antibodies against DevBCA, TolC:
- prepare 1:5000 dilution in TBS-T
- incubate at 4 °C overnight under gentle shaking
Washing
Washing
- incubate in TBS-T for 3 x 15 min at room temperature
Secondary antibody
Secondary antibody
- prepare 1:5000 dilution of anti-rabbit antibody
- incubate for 1 hour at room temperature
Washing
Washing
- incubate in TBS-T for 3 x 15 min at room temperature
- Detection kit: chemiluminescent detection of HRP-conjugates was performed with the “Clarity Western ECL” kit (Biorad)
- Detection system: ChemiDoc XRS system (Biorad) with cumulative image recording using the Quantity One software (Biorad).