Bradford Protein Assay - (Apr/07/2001 )
We are having problems probing the 18S rRNA gene in the rat liver. Total genomic DNA has been isolated. PCR amplification of a 643 base fragment has been successful. There is no reason to assume that the labelling has not worked either. Genomic DNA was restricted first before being run out on an agarose gel. Everytime we use these methodologies to look for genes encoded by mitochondrial DNA we are successful but when we look at nuclear DNA we do not get bands (3 times thus far). Why ???
Coomassie Brilliant Blue R250 (or G250) is used in the Bradford's dye binding method. I understand that you are quantitating the protein eluted from the gel filtration column. The dye binding method is sensitive and depends on the acidic strength of the dye and the availibility of a particular form of the dye (i.e., red form). The pH should be very low. I used to get linear curve for the standards in the range 1 ug to 40 ug in the total volume of 2 ml.(For details see a manuscript "Shareef and Shetty, August issue of Anal. Biochem, 1998). There is no enzymatic reaction involved here as far as i know. Good luckMomin