SDS-PAGE and Western blotting

Simple animated videos in the slides to explain SDS-PAGE and Western blot easily. SDS and Western blot protocol and reagent composition

1. SDS PAGE western blot

2. Main steps Sample preparation Electrophoresis (SDS-PAGE) Transfer of protein and staining (Western blot)

3. Reagents

4. SDS-PAGE Reagents 30% Acrylamide Acrylamide 29.1 gm Bis acrylamide 0.90 gm ( Dissolve in 60 ml MQ and make up to 100 ml ) Stacking Gel Tris Buffer (pH-6.8; 1.25 M) Tris Base 15.12 gm Dissolve in 60 ml MQ, adjust pH to 6.8 and make up the volume to 100 ml . Separating Gel Tris Buffer (pH-8.8; 1.8 M) Tris Base 22.72 gm Dissolve in 60 ml MQ , adjust pH to 8.8 and make up the volume to 100 ml.

5. Reagents 10% SDS PAGE 12% SDS PAGE 15% SDS PAGE Distilled water 3.9 ml 3.3 ml 2.3 ml Tris ( pH-8.8) 2.5 ml 2.5 ml 2.5 ml Acrylamide 30% 2.40 ml 4 .0 ml 5.0 ml 10% SDS 100 µ l 100 µl 100 µl 10% APS 100 µl 100 µl 100 µl TEMED 10 µl 10 µl 10 µl Distilled water 2.7 ml Tris ( pH-6.8) 375 µl Acrylamide 30% 600 µl 10% SDS 37.5 µl 10% APS 37.5 µl TEMED 10 µl Resolving/Separating Gel (1.5mm) Stacking Gel (1.5mm)

6. Gel percentage depend on the size of protein

7. Tris (pH = 6.8; 1.25 M) 0.5 ml SDS 0.2 gm β-Mercaptoethanol 0.5 ml Bromophenol blue 1.0 mg Glycerol 1.16 ml Tris 3.03 gm SDS 1.0 gm Glycine 14.4 gm Sample Buffer (5x) for 10 ml Running Buffer (pH – 8.0) Coomassie brilliant blue dye 0.1% Methanol 50% Glacial acetic acid 10% Water 40% Coomassie brilliant blue stain

8. De-stain solution Methanol 50% Glacial acetic acid 10% Water 40% Procedure to stain the gel Keep the gel in the CBB stain solution for 2-4 Hrs. on rocker. Then transfer the gel in the de-stain solution till the blue color bands are visible clearly without dark background . Coomassie brilliant blue stain Silver stain

9. Transfer of protein 1X Transfer buffer ( 1 L) Glycine (192mM) 14.4g Tris base (25mM) 3.02g Methanol (10%) 100ml Water 900ml Wash buffer (PBS-T) Phosphate buffer saline (PBS) 1X 1000ml Tween-20 0.05%

10. Step 1 SAMPLE PREPARATION

11. Lysis buffer Protease and phosphatase inhibitor Preparation of lysate from cell culture Preparation of lysate from tissue Determination of protein concentration Preparation of samples for loading into gel Requirements

12. Protein location and lysis buffer choice

13. Electrophoresis Step 2

14. Preparation of PAGE gel Positive control Molecular weight markers Loading sample and running of gel Use of loading controls Requirements

15. Apparatus for SDS-PAGE

16. SDS gel preparation Components 30% ACRYLAMIDE (Acrylamide + bis -acrylamide) TRIS BASE (pH 6.8 & 8.8) 10% SDS TEMED APS (Ammonium per sulphate ) WATER

17. Sample loading and Gel run Gel running buffer SDS GLYCINE TRIS BASE Water

18. Loading controls

19. Transfer of protein and staining (Western blot) Step 3

20. Visualization of protein in gel Transfer Visualization of protein in membrane: Ponceau red Blocking the membrane Incubation with primary antibody Incubation with secondary antibody Development method Steps Wash with 1X PBST Wash with 1X PBST Wash with 1X PBST Wash with 1X PBST

21. Requirements Nitro cellulose membrane Blotting sheet Transfer buffer Wash buffer (PBS-T) Blocking buffer 1 and 2 antibodies HRP substrate Detection reagent Ponceau Red 1g Acetic acid 50ml Water 950ml Ponceau Red

22. Apparatus

23. Transfer protein from gel to membrane

24. Results SDS PAGE Western blot