How to increase Western blot sensitivity? - (Aug/19/2006 )
I had a primary antibody from hell (nothing showed up, whatsoever)... then i ran another western, left the primary on overnight... got sick... so the thing was left for 3 days. decided to finish the experiment anyway... it was the best western ever, a beautiful signal.
i use a 1 in 50 000 dilution for my secondary. with the ECL system, if you put too much secondary on, the film is reversed (ie white lines where there should be dark ones).
i do a 2 hour shaking with the secondary, but that's because i'm following a cook book, and won't question it (it works for me).
if you use film, you could try to expose it for longer. if you use the fancy imager , check out the software that comes with it, maybe the software can be more picky about which signals it picks up.
if you're getting signal with the positive control (is it a nice strong band?), and nothing else, perhaps it the lysates. are they fresh? do they have protease inhibitors? is there enough protein in each well?
don't get too depressed.
the second one strangly enough no detection of any bands except the positive control.
my professor said its not recommended to use 1:5000 dilution of second antibody in ECL detection system?
and i am already using HRP antibody
i am going to do it again , wish me luck.
It seems that your conditions are fine. Your professor is right, several second antibodies have to be diluted a lot, even if the provider says that you should use it at 1:5000 (they want to sell more ).
what do you use as positive control? is it a purified protein, a cell extract? what the amount you loaded? It could help to understand why you see nothing from your samples.
my problem is
i did the same gel, same membrane ( cut into half) same first AB diltuion and only i was comparing between 2 concentrations of second AB ( 1:5000 for one hour and 1:50000 for 4 hours)
the samples were the same run in duplicate 10 micron each well( i have 3 samles induplicate and one positive control one negative control and one all blue marker so its 9 wells out of 10 used.
my positive control was my total cell lyste extraction to be sure that my protein is there in the other samples.
going crazy really!!!!!!
a quick question if i am using total cell lysate as a positive control why do i get a nice band with the positive control and not with the samples?
my prof said we can try to change the lysis buffer but i dont get it as the positive control is fine???