Trying Bradford test from scratch

The previous experiments using the commercial products where not that convincing and we are not sure why.

We therefore decided to repeat the original experiment using commercially available products:

• BSA (5g on ebay)
• Coomassie Brilliant Blue G250 CI42665 Acid Blue 90 (5g on ebay)

And based on the original publication from Marion M. Bardford

The current version of the spectrophotometer only allows 3mL cells and from the protocol we will therefore use the Protein assay (standard method).

For the standard protocol we need:

• 10 to 100µg of protein in 0.1 mL (NaCl 0.15M) (0.1g to 1g per liter)
• 5mL protein reagent

Solution of NaCl 0.15M

500 mL of NaCl 0.15M: 4.38 g to 250 mL

Protein solutions

We prepare 10mL of BSA solution: 10mg in 0.15NaCl (1g/L)

Bradfort reagent

• 100 mg of Coomassie Brilliant Blue G-250 in 50mL ethanol 95%
• 100 mL 85% (w/v) phosphoric acid
• complete to 1L

We will prepare 50mL of the reagent but we have only H2SO4 2N

• 4 mg of oomassie Brilliant Blue G-250 in 5mL ethanol 95%
• 10 mL of H2SO4 1M (2N)
• bring to 100mL with water

note

update experiment bradford

Experiments

Solutions:

• A: BSA 1g/L (in NaCl 0.15M solution)
• B: NaCl 0.15M
• C: Bradfort reagent

Exp	A	B	C	R	G	B
1	0.00	0.10	5
2	0.02	0.08	5
3	0.04	0.06	5
4	0.06	0.04	5
5	0.08	0.02	5
6	0.10	0.00	5