Problem assessing protein concentration
Posted 15 March 2009 - 07:41 AM
I am wondering whether you can help me,
I am attempting protein purification on Nickel beads from 293-T cells culture medium, therefore the amounts of the obtained protein are relatively small, though after concentration and SDS-PAGE, followed by coommassie staining I am able to see a a very band, which should correspond to about 200-500 ug/mL,
BUT when I am trying to determine protein concentration by various well known techniques such as BCA, bradford and even fluorescamine, the signal doesn't go beyond background (PBS in my case). This doesn't make any sense, since the band which appears on the gel is big enough to be detected! I am wondering whether imidazol which i am using for protein elution may be the reason for that, eventhough the final protein which i am working with is dialysed against PBS and should not contain imidazol.
What might be the reason for that lack of protein in all these assays while I can see the band in the SDS-PAGE gel?
thank you very much in advance.
p.s. I am new to this forum so excuse me if something I am written is not according to the accepted.....
Posted 15 March 2009 - 08:02 PM
1. DO the standards show up in the assay....Is the kit working fine?
2. There is somethign in yr buffer which gives a high signal.
If its really urgent, go for densitometry.....just run yr protein with known concentrations of standard BSA, stain/destain the gel and quantify under a gel documentation system. Draw a standard curve between concentration of standards and their quantification value. Use this curve and the quantification value of yr protein to estimate its concentration.
Hope it helps
Posted 15 March 2009 - 08:04 PM
Posted 16 March 2009 - 08:42 AM
even with salt present you may lose a significant amount of protein when working with low concentrations.
test the tubing for the protein by taking a piece and boiling it with 1x laemmli sample solution and run the eluate on a gel.
you may be able to minimize the loss by using a spin concentrator and exchange the buffer by diafiltration.
genius does what it must
i do what i get paid to do
Posted 18 March 2009 - 12:53 AM