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Solutions

Western Blot Solutions

Ponceau Red Stain, (500 ml)
0.5% Ponceau Red; 1% Acetic Acid
  • 2.5 g Ponceau Red
  • 5 ml glacial acetic acid
  • To 500 ml with dH2O
3% Milk and 2% BSA in TBS or T-TBS to block membranes after transfers, (100ml)
  • 3 g Milk
  • 2 g BSA
  • 1 ml Thimerosol
  • To 100 ml with TBS or T-TBS (T-TBS is TBS with 0.05% Tween)
1x Alkaline Phosphatase Buffer (AP), (1L)
Note: Make in plastic bottles as might come out of solution otherwise. This doesn't keep well. The 10x stock keeps fine. If we have the 10x stock, combine 100 ml of the stock with 900 ml Oasis water.
  • 20 ml 5 M NaCl
  • 5 ml 1 M MgCl2
  • 100 ml 1 M Tris pH 9.5
  • To 1 L with dH2O
  • (Final concentrations of 100 mM NaCl; 5 mM MgCl2; 100 mM Tris)
Reference: Antibodies: A Laboratory Manual, page 407.

10x Alkaline Phosphatase Buffer (10x AP), (1L)
  • Note: Make in plastic bottle; use a deionized, autoclaved bottle of water.
  • Start with about 700 ml water
  • Add 58 g NaCl (fw 58 g)
  • 121 g Tris base (fw 121 g)
  • 50 ml 1 M MgCl2 (on solutions shelf)
  • pH to 9.5 using NaOH or HCl
  • Adjust volume to 1000 ml
Semi-dry transfer to nitrocellulose
For MS39, use nitrocellulose and the following solutions:
  • Anode Buffer #1: 0.3 M Tris, 10% methanol, pH 10.4
  • Anode Buffer #2: 25 mM Tris, 10% methanol, pH 10.4
  • Cathode Buffer: 25 mM Tris, 40 mM e-amino-n-caproic acid, 20% methanol, pH 9.4
    There are 1 M stock solutions of Tris, pH 9.4 and Tris, pH 10.4 or 10.5 which can be used.
Anode Buffer 1 (10% MeOH; 0.3 M Tris) 100 ml
  • 30 ml 1 M Tris, pH 10.5
  • 10 ml MeOH
  • 60 ml dH2O (very good water)
Anode Buffer 2 (10% MeOH; 25 mM Tris, pH 10.5) 100 ml
  • 2.5 ml 1 M Tris, pH 10.5
  • 10 ml MeOH
  • 87 ml dH2O (very good water)
Cathode Buffer (20% MeOH) 100 ml
  • 1.25 ml 1M Tris, pH 9.4
  • O.26 g e-amino-n-caproic acid (also called 6-amino-hexanoic acid) (should be filed under "A")
  • 20 ml MeOH
  • 78.5 ml dH2O (very good water)
For most other transfers to nitrocellulose using the semi-dry blotter
(Check manual for other recommendations)
  • Anode Buffer #1: 0.3M Tris, 10% methanol, pH 10.4
  • Anode Buffer #2: 25 mM Tris, 10% methanol, pH 10.4
  • Cathode Buffer: 25 mM Tris, 40 mM 6-aminohexanoic acid, 10% methanol, 0.05% SDS
Semi-dry transfer to PVDF
  • Omit the methanol in the anode buffers and the cathode buffer.
  • Add SDS to 0.1% in the cathode buffer.
  • Wet the PVDF membrane in methanol.
  • When saturated, switch to very pure dH2O.
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Last update Oct 2, 2000
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