General Solutions
NOTE: For a listing of many reagents required to prepare the following solutions, see Standard Laboratory Chemicals.
Blotto A (for general use):
Blotto B (for use with anti-phosphotyrosine antibodies):
- 1x TBS (sc-3715), 1% milk, 1% blocking reagent, 0.05% Tween-20 (sc-29113).
NOTE: In some cases, milk may be left out entirely, but this will result in somewhat higher backgrounds. - Available pre-made (sc-2335).
- For all phospho-specific antibodies: Add 0.01% (v/v) of each Phosphatase Inhibitor Cocktail A and B (sc-45044 and sc-45045) to inhibit phosphatase activity.
Diaminobenzidine Tetrahydrochloride (DAB):
- Dissolve 5 mg DAB (sc-24982) in 100 ml 100 mM Tris-HCl, pH 7.6 (sc-216106), and add 0.1 ml 0.3% hydrogen peroxide (sc-203336).
- Prepare fresh DAB solution daily.
Electrophoresis Sample Buffer (2X):
- 100mM 2-(N-Morpholino)-ethanesulfonic acid(MES) (sc-216094), 10 mM Na EDTA (sc-29092), 15% glycerol (sc-29095), 1.5% SDS (sc-24950), 0.3% Triton X-100 (sc-29112), 100mM TCEP-HCL (sc-203290), 7.5 mM DTT (sc-29089), 0.0025% Bromophenol Blue (sc-24971).
- Available pre-made (sc-24945).
Phosphate buffered saline (1x PBS):
- 9.1 mM dibasic sodium phosphate (sc-203277), 1.7 mM monobasic sodium phosphate (sc-301829) and 150 mM NaCl (sc-203274).
- Adjust pH to 7.4 with NaOH (sc-301824).
- Available pre-made in liquid (sc-24946) and powder (sc-24947) forms.
RIPA Buffer:
- 1x TBS (sc-362185), 1% Nonidet P-40 or Igepal CA-630 (sc-280818), 0.5% sodium deoxycholate, 0.1% SDS (sc-24950).
- This may be made in large volumes. Add inhibitors at time of use from the following stock solutions.
- Available pre-made (sc-24948).
Subbing Solution:
- 0.3% (w/v) gelatin (sc-218567), 0.05% chromium potassium sulfate in distilled H2O.
Tris Buffered Saline (1x TBS):