Chemical Forums

Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: macdonda on June 24, 2005, 08:03:38 AM

Title: How to determine protein presence?
Post by: macdonda on June 24, 2005, 08:03:38 AM

Hi, I'm looking for a relatively basic method to determine if a sample contains a significant amount of protein.  I have thought of Kjeldahl for total nitrogen or via a.a.  Is there a simpler method?  Is there an enzyme that would break the proteins up and then I could test for specific amino acids?

Thanks

Title: Re:How to determine protein presence?
Post by: arnyk on June 24, 2005, 06:27:59 PM

If protein is present:

Sample + Benedict's + Heat = Purple

Sample + Biuret + Heat = White precipitate

I don't know how far you can go with that, but that's just basic detection.

Title: Re:How to determine protein presence?
Post by: savoy7 on June 25, 2005, 10:39:29 PM

If you want to quantify protein and you have a spec:

1) Bradford Dye - pretty easy to do and cheap

some other ones - I've not used just done once a long time ago
2) Lowry assay
3) Smith assay


If you have a good spec - you would need it to have UV capabilities and a quartz curvette.

A₂₈₀ = 1 A (mL/cm mg) x [Conc.] (mg/mL) x 1 (cm)

Using UV absorption can be tricky - has to be a pure sample (free from other UV absorbing substances) and different proteins will absorb differently.  Some people use A₂₀₅.  


NOTE:
The Bradford assay is just using Coomassie Blue dye.  It is okay but has problems with detergents and sometimes can be tricky due to the proteins.  For example, one lab I was in used a animal protein for it's standard curve and we were testing for levels of a plant protein.  That's not the best.  For the best results, standard and test variable should be the same.


The Lowry and smith assays are both good, but a little time consuming to set up.  I also think they are time dependant (can't remember ).