- 1X Cell Lysis Buffer:
20mM Tris-HCl, pH 7.5, 150mM NaCl, 1% NP-40,2 mM EDTA, 1µg/ml leupeptin, 1µg/ml aprotinin ,1mM Na3PO4, 1mM PMSF, 5mM NaF, 3mM Na4P2O4 - 5X SDS Sample Buffer:
312.5mM Tris-HCl (pH 6.8) , 10% SDS (w/v), 250mM DTT, 50% Glycerol, 0.05% Bromophenol Blue (w/v) Use at 1X, 80.0g NaCl, 4.4g Na2HPO4, 2.4g KH2PO4, 2.0g KCl. Add ddH2O up to 10L, pH to 7.2 with HCl - 10X SDS Running Buffer:
Dissolve 144g of Glycine, 30g of Tris base and 10g SDS in 800ml of distilled H2O. Add distilled H2O to 1 liter. Use at 1X - Transfer Buffer:
3.0g Tris base, 14.4g Glycine 200ml Methanol. Add distilled water to 1.0L | - 10X TBS-T (Tris-buffered saline containing Tween-20): Dissolve 80g of NaCl, 2g of KCl, 30g of Tris base and 10ml, Tween-20 in 800ml of distilled H2O. Adjust the pH to 7.4 with HCl. Add distilled H2O to 1 liter. Use at 1X (containing 0.1% Tween-20).
- Blocking Buffer:
1X TBS-T with 5% nonfat dry milk - Wash Buffer: 1X TBS-T
- Direct-Blot™ Antibody Dilution Buffer: 1X TBS-T with 5% nonfat dry milk. **If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk.
- Alternate Blocking Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA)
- Alternate Direct-Blot™ Antibody Dilution Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA)
- Blotting Membrane: Nitrocellulose or PVDF membrane
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