Materials
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1. Stock acrylamide solution Acrylamide 30% - 30.0 g Bisacrylamide 0.8% - 0.8 g Water to - 100 ml 2. Separating gel buffer (pH 8.8) 1.875 M Tris – HCl - 22.7 g Water to - 100 ml 3. Stacking gel buffer (pH 6.8) 0.6 M Tris – HCl - 7.26 g Water to - 100 ml 4. Polymerizing agents a. Ammonium persulfate, 5% - 0.5 g in 10 ml distilled water (prepare fresh) b. TEMED – Fresh from the refrigerator 5. Electrode buffer (pH 8.2 – 8.4) 0.05 M Tris - 12.0 g 0.122 M Glycine - 28.8 g 0.2% SDS - 2.0 g Water to - 2 L (This may be used 2-3 times) 6. Sample buffer (5X concentration) Tris –HCl, buffer pH 6.8 - 5.0 ml SDS - 0.5 g Sucrose - 5.0 g Mercapto ethanol - 0.25 ml Bromophenol blue - 1.0 ml (0.5% w/v solution in water) Water to - 10 ml (Store frozen in small aliquots. Dilute to X concentration and use.) 7. SDS solution 10% - Store at room temperature 8. Standard marker protein PROTEINS MW (in Daltons) a - lactalbumin 14,200 Trypsin inhibitor soybean 20,100 Trypsinogen 24,000 Carbonic anhydrase 29,000 Glyceraldehyde –3 phosphate dehydrogenase rabbit 36,000 Egg albumin 45,000 Bovine albumin 66,000 Dissolve the above proteins in single strength sample buffer at a concentration each of 1 mg / each ml. Load the well with 25-50 m l. 9. Protein stain solution Coomassic brilliant blue R250 - 0.2 g Methanol - 40 ml Acetic acid - 10 ml Water - 50 ml First dissolve the dye in methanol and proceed. Use fresh preparation every time. 10. Destaining solution As above without the dye |