I am detecting a protein from a kind of samples. For positive concol, I layer indicated protein in different amounts as 10ng 50ng and 100ng on the gel. But the strange is sometimes I got blot on 50ng and 100ng after developing on film, however, sometimes I didnt get any. The following is the infromation could be useful for your valuable answer.
1) the MW of target protein is 28kD
2) We have two kinds of the purified protein for positive control.
Rat brain sample (we know that it release the target protein, and result is positive.)
Bovine-origined target protein (Results negetive, even in 100ng, but before me other people got positive results, and I also got positive result in 100ng dose before these days.)
3) Primary antibody is from rat.
4) Secondary antibody is Pod-goat anti-rat IgG
5) Sample is prepared by SB with 2-ME and boil for 5 min.
We guess probably this problems happend because the protein we used for positive control come form bovine, well the primary and secondary antibody come from rat. But we compared the struture of the two kinds of protein, 99% same.
Need your answer.