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.
1. 10 mg/ml PMSF (sc-3597) in isopropanol (add at 10 µl/ml RIPA)
2. Aprotinin (sc-3595) (add at 50 KIU/ml RIPA)
3. 100 mM sodium orthovanadate (sc-3540) in frozen aliquots (add at 10 µl/ml RIPA)
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):