Nuclear protein concentration-BCA
A. Samples used for EMSA
DNA/RNA binding proteins in
samples used for EMSA must be, 1) native,
2) activated 3) at detectable level. The levels of a specific binding
factor from deferent samples can only be compared if
an equal amount of
proteins for all the samples are applied to EMSA.
Unfortunately, we found that the concentration of nuclear proteins measured
by many of our customers is not reliable.
B. Common problems for protein concentration determination.
(1) The range of standard curve for protein concentration used for Western Blot is too high for determine nuclear protein concentration. Protein concentration of samples for Western blot is usually at 10-30 ug/ul, but protein concentration of nuclear extracts is only around 1-3 ug/ul. If a measurement results in a difference of 2ug/ul protein, it is not a big issue for Western blot, but would be a serious problem for nuclear proteins used for EMSA.
(2) Low protein level of nuclear extracts needs am accurate measurement. Comparing to other detection methods such as Bradford, Lowry and Folin-Phenol, the BCA-based method is recommended for the determination of nuclear proteins concentration due to its high repeatable and consistent results, if the interferers is avoided.
(3) Some protein extraction kit on the market contains EGTA, DTT etc. which may cause the color change in the detecting solution without proteins and result in high O.D. reading. False high protein concentration could result in the failure of EMSA.
(4) Some nuclear extraction kits, according to its operation manual, are easy to induce the contamination of cytoplasmic proteins. As the proteins in cytoplasm is tens-fold higher than that of nuclear proteins. Minor contamination of cytoplasmic proteins into nuclear extracts could increase protein concentration significantly, but reduce the ratio of DNA/RNA binding protein factor in the extracts greatly.
C. Viagene developed a modified BCA kit for detecting nuclear protein concentration. The kit would provide a reliable result when used with Viagene's nuclear protein extraction kit.