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Comparative Physiology @ Guelph

Protein Concentration Assay

Pierce Coomasie Kit

Working range with BSA 125-1000ug/ml


  • Dilute unknown at least 1:1 with water, can do 1:5 1:10 etc
  • Do not dilute standards
  • Pipette 50ul each standard or unknown sample into labeled test tube
  • Do standards and unknowns in duplicate
  • Gently mix contents of reagent by inverting bottle several times
  • Add 1.5ml Coomassie Plus Reagent to each tube, vortex
  • Let stand at room temp 10 minutes
  • Set spectrophotometer at 595nm, measure all samples
  • Subtract the (average) 595nm blank value from all other values
  • Prepare standard curve, linear regression

One example of Standards

Vial Volume Diluent Volume BSA Final Conc Stock=2000ug/ml

A 0 300ul stock 2000ug/ml

B 125ul 375ul stock 1500ug/ml

C 325ul 325ul stock 1000ug/ml

D 175ul 175ul A 750ug/ml

E 325ul 325ul B 500ug/ml

F 325ul 325ul E 250ug/ml

G 325ul 325ul F 125ug/ml

H 400ul 100ul G 25ug/ml

I 400ul 0 0 Blank

  • You can also do a serial dilution 2000, 1000, 500, 250 125 62.5
  • Note: Can also add 66.67ul sample plus 2 ml Coomassie reagent