TITLE: SDS PAGE & Western Blot for Membrane proteins
AUTHORS: Dennis Dienst

[DESCRIPTION]
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[STEPS]
?. [Prepare 4 x loading dye]
For 10 mL:                                       final conc.     617 mg DTT                                      400 mM1,25 mL 2M Tris-Cl (pH 6.8)           250 mM4 mL Glycerol                                   40% (w/v) 4 mL 20% SDS                                    8% (w/v)ad     H2O to 10 mL
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.
?. [Sample denaturation]
adjust protein samples to equal concentrations by adding PBSadd 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 e.g.: 2.5 µL LD + 7.5 µL sample   4 µL LD +  12 µL sample   5 µL LD +  15 µL sample
Do not boil, in order to prevent aggregation !
50 °C
?. [Gel loading]
spin down denatured samples for 1 min at 12000 gassemble 'Mini-Protean' chambers and add a mock plate if requiredfill 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 pipetteload 4 µL 'PageRuler prestained Protein ladder' into one well
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]
?. [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 lidinsert cassette into the instrumentTransfer conditions:Program 'Standard SD' - 30 min (up to 1.0 A; 25 V constant) http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10016505D.pdf
?. [Staining]
?. [Blocking]
cf.: Analytical Biochemistry 426 (2012) 22–23 (link below) https://doi.org/10.1016/j.ab.2012.03.028
?. [Washing/ Equilibration]
?. [Primary antibody]
for antibodies against DevBCA, TolC:prepare 1:5000 dilution in TBS-Tincubate at 4 °C overnight under gentle shaking
?. [Washing]
?. [Secondary antibody]
prepare 1:5000 dilution of anti-rabbit antibodyincubate for 1 hour at room temperature
?. [Washing]
?. [Prepare TBS-T buffer]
Stock solutions:1) 1 M Tris (pH 7.5) - for 1 L: 121,14 g Tris, titrate with HCl2) 5 M NaCl - for 500 mL: 146,1 g NaClFor 1 L TBS-T:                               100 mL 1 M Tris (pH 7.5)  30 mL 5 M NaCl    1 mL Tween 20
When using concentrated HCl (37%), work in the hood! HCl 'smoke' attacks your lungs when inhaled!
?. [Washing]
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).