Biology Lab Help – Protein Assays
February 1, 2010 at 10:08 pm #12698ZabuliusParticipant
Finished all parts of my lab and report except this final question. I’m not quite sure I fully understand the concept of why it happens.
Most proteins have an absorption maximum at 280 nm. If that is so, why do you assess the absorbance in the Bradford assay with the wavelength set at 595 nm? (2 reasons)
Any help or insight would be greatly appreciated.
February 2, 2010 at 12:56 am #97190MrMisteryParticipant
do you know how a bradford assay works?
February 2, 2010 at 4:20 am #97198ZabuliusParticipant
Yes, atleast I believe so. It involves the dye Coomassie Blue. The dye binds to both basic and aromatic amino acids when reacted with a protein sample. I also know the absorbance of the dye shifts when mixed with a protein sample but other than that I can’t say much.
February 2, 2010 at 7:40 am #97200JackBeanParticipant
The one and only reason is, that the complex protein-Coomassie has maximum at that wavelength
February 3, 2010 at 5:37 pm #97233clevermizoParticipantquote Zabulius:
Why Bradford over A280?
Well, A280 is fine if you are working with a purified protein sample. However, very often you would like to measure the total protein concentration of a lysate, a homogenate or any other preparation in which there is a mixture of components. Any UV-absorbing components will add to the background, and these preparations often have a lot of these things. There will be too much error in the A280 measurement. You won’t know how to subtract the background from the protein. Thus a different assay like the Bradford assay is useful. The Bradford assay is also less sensitive to chemicals used in sample preparations (like lysis buffer components, though too much SDS can screw it up).
If I purify protein by some sort of affinity purification (GST, 6xHIS, immunoprecipitation, etc), I can often measure concentration of the eluates with A280 and that works fine.
The reason why you measure A595 is that absorbance at A595 correspondents to bound Coommassie/Protein complexes (blue color). Therefore, A595 is proportional to the amount of bound complex, and can therefore be used as a measure of total protein content, if it is assumed that protein binds Coommassie across all samples with the same ability.
I’m not sure if the question is asking anything more than these two points.
- You must be logged in to reply to this topic.