| • |
Blotto A (for general use): 1x TBS, 5% milk, 0.05% Tween-20. Available pre-made (sc-2333). |
| • |
Blotto B (for use with anti-phosphotyrosine antibodies): 1x TBS, 1% milk, 1% BSA, 0.05% Tween-20. 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 in 100 ml 100 mM Tris-HCl, pH 7.6, and add 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB solution daily. |
| • |
Electrophoresis sample buffer (2X): 100mM 2-(N-Morpholino)- ethanesulfonic acid(MES), 10 mM Na EDTA, 15% glycerol, 1.5% SDS, 0.3% Triton X-100, 25mM TCEP-HCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Available pre-made (sc-24945). |
| • |
Phosphate buffered saline (1x PBS): 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate and 150 mM NaCl. Adjust pH to 7.4 with NaOH. Available pre-made in liquid (sc-24946) and powder (sc-24947) forms |
| • |
RIPA buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add inhibitors at time of use from the following stock solutions. Available pre-made (sc-24948).
| 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) |
|
| • |
Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled H2O. |
| • |
Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 7.4; 150 mM NaCl (Tris: cat # sc-3715). Available pre-made in liquid (sc-24951) form. |
 |
|
|
|
|
