Principles of Biochemistry (2+1)

Reagents

     1. Stock acrylamide solution: Acrylamide 30% - 30.0 g, bisacrylamide.0.8% -0.8 g, Water - 100 ml

2. Separating gel buffer: 1.875 M tris – HCl - 22.7 g, pH 8.8, Water - 100 ml

3. Stacking gel buffer :0.6 M tris HCl - 7.26 g, pH 6.8, Water - 100 ml

4. Polymerising agent:

          Ammonium persulphate 5% -0.5 g/10ml (Prepare fresh before use)

          TEMED – fresh from the refrigerator.

5. Electrode buffer: 0.05 M tris - 12.0 g, pH 8.2 - 8.4 - 0.192 M glycine - 28.8 g, 0.1% SDS - 2.0g, Water - 2.0 lit. No adjustment required (To be used 2-3 times)

6. Sample buffer: 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 - 10 ml Store frozen in small aliquotes.

Dilute to I-X concentration and use. Sodium dodecyl sulphate 10% solution – store at room temperature

7. Standard marker proteins

Protein	MW daltons
Lactalbumin	14,200
Trypsin inhibitor - soybean	20,100
Trypsinogen	24,000
Carbonic anhydrase	29,000
Gelyceraldehyde  3                  phosphate      dehydrogenase - rabbit	36,000
Albumin - egg	45,000
Albumin - bovine	66,000

 

        Dissolve the above proteins in single strength sample buffer at a concentration of 1 mg per ml. Load the well with 25-50 ml.

 8. Protein stain solution: Coomassie brilliant blue Rs. 250 - 0.1 g, Methanol - 40 ml; Acetic acid - 10 ml Water - 50 ml. First dissolve the dye in methanol and proceed. Use fresh preparation every time.

9. Destaining solution : As above without the dye