many factors can play a role such as optimization of the primary and secondary antibody concentration, TMB, incubation time, voltage, and protein concentration.
many factors can play a role such as optimization of the primary and secondary antibody concentration, TMB, incubation time, voltage, and protein concentration.
I especially agree with protein concentration and primary antibody dilutions.
Take into consideration the abundance of the protein of interest within your sample, if it is glycosilated and the origen/treatment of your sample. Depending on this factors you will need to vary the amount of sample to use in your western, the primary antibody concentration, the % of acrylamide of your SDS-PAGE and the voltage of the electrophoresis. These last two issues are important if your protein is glycosilated because the different patterns of glycosilation can cause a thick tending to smear band which corresponds to the different weights contributed by the glycosilation patterns of the protein.
Hi, Reena, Your question is too genaral, it is difficult to answer precisely. What do you have so far? Only not sharp band, or high backgound or no band? Do you have a good positive control?If your positive control is not shrap, it mean there is a problem with running of loading. You should,exactly follow the protocl in the term of pH, acrylamide concentration etc. I would suggest to use pH of the separation gel 9.1. In this case yopu will have more sharp bands. But alos voltage, current, buffer is importnat as well.
Good luck!
I do get bands in my blots. Bt i have high background signals.. And im working on dose dependant samples bt it is very tough to get a nice dose dependant increase of band width often the bands are in odd shape
Reena, for removing background you have to play around with your antibody dilution and increase the washes (time and number). You can also increase wash stringency by slightly increasing the detergent component (e.g. tween), For sharper bands, check pH of all the gel and buffer components as suggested above. Also, running the gel slower keeping the gel running tank cool will help prevent smiling or crookedness of bands.
Hi, Reena, thanks for clarifications! you should increasing duration of the blocking (milk or BSA), minimize agination during primary antibody incubation and increasing washing step, may be even with more deetregent in the washing solutions. Also, you can try increasing amount of protein per track. Good luck!
Use gradient PAGE gels. They compress the protein bands more than homogeneous PAGE.
What kind of transfer are you doing? I find that some of the "quick transfer" methods that Invitrogen and Bio-Rad offer sometimes have a high background when you're doing longer exposures. For these, I went back to doing an old-school wet transfer and it solved the problem.
I do wet transfer at 100 voltage for 2 hours. I make sure that the tank doesn't get hot throughout the transfer process. I wash with 0.1% tween 20 for 5 minutes thrice.
Not to forget the final detection i incubate the blot for ten minutes with the chemiluminescent as per the protocol. is it considered too long
That seems like a long transfer unless you are going after a high MW protein. So you might be pulling your protein right through the membrane and into the filter paper. Do you see ladder on the filter paper? I usually find that for proteins under 90kDa 1h at 100V is plenty. You may also want to try blocking in 5% milk or BSA before washing.
if u r getting lot of background ...den increase salt concentration in washing buffer ie. TBS with 0.1% tween-20...
u can increase the tween -20 conc upto 0.2%
wash for 4 times 15 mins each
Yes i see clear ladder michael. i was told previously to increase the transfer time when i increase the gel thickness .
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