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SDS-PAGE and Immunoblotting

Tim Corson, PhDIndiana University School of Medicinehttp://iueye.iu.edu/corson November 2010

SDS-PAGE

1. Prepare bis-tris gels or use precast TGX gels (Bio-Rad)2. Dilute samples appropriately in NuPAGE sample buffer (for bis-tris) or Laemmli

buffer (for TGX)

3. Boil samples 5 minutes at 100C in heating block4. Load 10-50 L protein sample per well5. Add appropriate running buffer to tank6. Run gel 200 V, 0.5 hour7. Remove gel from glass or plastic plates, cut off stacking gel

Transfer1. Soak 69 cm nitrocellulose membrane in transfer buffer appropriate for gel

system (use forceps!)

2. Prepare transfer sandwich: soak sponges in buffer, layer a buffer-soaked blottingpaper sheet (710 cm) on top, roll out bubbles with a large test tube

3. Layer gel on top of paper, roll out bubbles4. Carefully place membrane on top of gel5. Layer another soaked blotting paper square on top, roll out bubbles6. Add sponge7. Close sandwich, place in transfer unit, with membrane side closest to positive

electrode (red)

8. Transfer 75 V, 90 minStaining

1. After transfer, gels and blots may be stained2. Shake gel in 5-10 mL Coomassie Blue (recyclable!) for 10 minutes, followed by

two 15 minute washes with water to destain, leaving dark blue protein bands

3. To stain membrane: Shake in 5 mL Ponceau S (recyclable!) for 5 minutes,4. Rinse thoroughly with water to visualize pink protein bands5. Cut or mark gel bands as necessary now; stain will disappear during blocking

Immunoblotting

1. Block membrane by shaking in 5 mL 5% skim milk powder in TBST, 1 hour2. Replace blocking solution with a similar solution containing an appropriate

dilution of primary antibody3. Shake for 1-3 hours at RT OR shake at 4C overnight (covered)4. Remove and save antibody solution (preserve with 0.02% sodium azide)5. Rinse blot twice with TBST6. Wash three times for five minutes in TBST7. Prepare secondary antibody dilution in 5% skim milk powder in TBST

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8. Add secondary antibody solution, incubate with shaking 1 hour at RT9. Discard antibody solution, and rinse blot twice with TBST10.Wash five times for five minutes in TBST11.Cover with ECL+ reagent (25 L Solution B + 975 L Solution A per blot)12.Transfer to GelDoc or film cassette (in Saran wrap) for imaging

RECIPES

2 Laemmli sample buffer

1514.2 mg Tris pH 6.8 (final 1 = 62.5 mM) 6 g SDS in 50 mL H2O (final = 3%) 40 mL glycerol (final =20%) 10 mL b-mercaptoethanol (final = 5%)

4 Running buffer for TGX Gels

For 2 L:

24g Tris base (final 1 = 25 mM) 115.2 g Glycine (final = 192 mM)

pH should be 8.3. For each gel tank, make 400 mL of 1 in H2O, add 4 mL (0.1%) 10%

SDS

Towbin Transfer buffer for TGX Gels

For 6 L:

18.15 g Tris base (final = 25 mM) 86.4 g Glycine (final = 192 mM) 1200 mL methanol (final = 20%) 4800 mL dH2O

20 MOPS/SDS Running buffer for Bis-Tris Gels

For 1 L:

209.2 g MOPS 121.2 g Tris base 20 g SDS 6 g EDTA (free acid) or 7.44 g disodium EDTA

20 NuPAGE Transfer Buffer for Bis-Tris Gels

For 500 mL:

40.8 g Bicine 52.32 g Bis-Tris 3 g EDTA (free acid) or 3.8 g disodium EDTA

When diluting to 1, include 20% (final) methanol