Protein determination - Old Skool style - I'm looking for a decent descriptive protocol for protein detection using th (Jun/22/2006 )

Hi all,

I have been away for a while ( ), and now I'm back!

I'm trying to put something of a small training module together to cover a range of basic lab techniques, and I also have to have a practical element to it.

What I want to do is something cheap and quite straight forward to do, which is get the trainee to use all of the techniques in a simple protein assay, and I've been asked to use Biuret, as it's quite cheap and straight forward, but I was hoping someone would be able to point me in the direction of a good protocol for this? Preferably making use of a microplate reader (we dont have a regular spectrophotometer for cuvettes etc).

Can anyone help?



Cheers,



HMilk

never done classical biuret (only know bca and thats +-expensive) but
check this

Biuret Protocol

and just scale down?

never done classical biuret (only know bca and thats +-expensive) but
check this

Biuret Protocol

and just scale down?

i have the protocols for biuret and microbiuret (i've used biuret for a lot of years) at work. i'll try to remember to give them to you when i am at work tomorrow.

Biuret Reagent and Assay:

dissolve in 500 ml diwater:
1.5 gm CuSO4-5H2O
6.0 gm NaKC4H4O6-4H2O (sodium potassium tartrate)

then, with mixing, add:
300 ml 2.5 N NaOH (10%)
(you may want to do this in front of the students, there is a striking color adjustment when added)

adjust to 1000 ml with diwater (we store at 4C indefinitely).

Assay:
add 4 ml biuret reagent to 1 ml sample+diwater. mix well. allow to sit for 20 minutes at room temperature. read at 550 nm.

prepare standard curve with 10 mg/ml BSA.

the assay is scalable. we use 0.2 ml sample with 0.8 ml reagent and standard curve to 2 mg (0.2 ml of the 10 mg/ml BSA standard), just maintain the 4:1 ratio of reagent to sample and adjust the standard curve accordingly (same concentration, lower amount).

hope this helps.