Which kit is more accurate for determination of protein concentration in SDS- Page sample buffer?
1- Protein assay kit (Bio-RAD)
2- 660 nm protein assay (Thermo scientific)
3- 2-D quant kit (GE healthcare life science)
Protein is extracted from lung biopsy and finally western blot will perform
Thanks
I like the BCA essay from Thermo because of it´s sensitivity of a wide range of protein concentration! I use tissue lysate (0.1 g of tissue in 1 ml of lysis buffer) and I can measure in a 96 well plate directly 3µl of lysate in a total of 250 µl of reaction. Only problem is its sensitivity to reduction agent.
The Bradford of Biorad is not suitable for high protein concentration, you need to add extra dilution steps in you protocol. But it is relatively cheaper that BCA, however it is prone to detergent artifact.
I do not know the 2-D quant kit.
Last comment, many actual equipment can measure protein concentration in a not colorimetric way (https://www.researchgate.net/post/Nanodrop_or_BCA_Bradford_assay)
If you had equipment for imaging gels (has to be hires camera, like in ImageQuant or sth similar) you could run SDS-PAGE of your samples with known standards (in range of concentrations), stain them with Ruby stain (BlueStain is not as good for quantitation) and compare the intensity of bands which should be proportional to the concentration.
Otherwise, no idea which one is better. You can also BCA protein kit, it is comapatible with SDS up to 5 %: http://www.piercenet.com/product/bca-protein-assay
Quantitation of protein by SDS-PAGE give you an estimate. The Lowry, BCA and Bradford methods are better for determine protein concentration. I recommend The Protein Protocols Handbook (John M. Walker), for choose a good method accord at your conditions.
@ Jazmin Alvarez: I would not say it is only an estimation, because you can get accurate results within 5 % error. I have quantified DARTs (parts of Antibody) using this technique, but with fluorescent dye.
It depends however what range of concentrations is handled and of course what stain, protein, software and camera is used. The tricky bit is background signal which might not be easy no extract.
However, that's true, that it might not be as accurate as other methods and it certainly needs more laborious procedures than simple protein assay. It is also less efficient.
Anyway, this is kind of off-topic.
Bradford is very sensitive to SDS, BioRad allow concentrations up to then 0.025%. BCA is better, Pierce allow up to 5%. I never tried the 660nm Protein Assay Kit but from the specs it looks good. Quantification after SDS-PAGE by fluorescence or densitometry is also doable although not trivial as you need a linear scanner. My 2 cents, given the options you raised try the 660nm.
Yoram
Just looked at the 660nm instructions, the 5% SDS limit is here as well, so BCA might be just as good...
See here: https://www.piercenet.com/instructions/2162070.pdf
with the Protein assay kit from Bio-Rad we didn't have any problem, but just remember to compare the protein concentration of different samples with this assay. Each assay can give you a slight change.
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