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2. BRADFORD ASSAY:
Use the Bio-rad Bradford reagent (stored in cold room).
NOTE: You should do standard curve with a known standard (BSA for most proteins, IgG for serum proteins for example) every time you are running a series of samples f or a Western. And you should run the standard curve against all samples being run on
Western ior the control, make up a solution of accurately measured out fraction V BSA in water at 1.4 mg/ml. Store at -20 C. Use glass tubes for the assay (not plastic!!!)
Use 20 ul of lysate from test samples when assaying for concentration.
Use the Standard Assay Procedure in the Bio-Rad Handbook, rather than the Microassay procedure (10-140 ug protein). Set up Elisa reader to x # of controls, under the point to point calculation program, reading at 595 wavelength.
1. In 7 tubes, set up controls as follows:
Standard BSA(ul) LysisBuff H20 Bradford Amt prot O.D.595
(ul) (ul) (ml)(ug) example
1 - 20 50 5 - 0
2 10 20 70 5 14 .147
3 20 20 60 5 28 .239
4 40 20 40 5 56 .341
5 60 20 20 5 84 .449
6 80 20 -- 5 112 .596
7 100 -- 5 140 .690
Sample Lysate LysisBuff H20 Bradford Amt prot O.D.595
1 20 80 5 ml
2 20 so .5 ml
3 20 80 5 ml
Let reaction sit for 5 minutes, mix well, and add 100-200 ul per well in 96 well titer dish going in this order with controls: Al-Hl, A2-H2 etc. Plate all samples in duplicate. The machine will
calculate your protein concentrations in your samples with the data from the standard controls.
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