Western Blot Background Troubleshooting

[Dianadlr]

My tip is:

Use a leveler to make sure the proteins are at same level.

[kevin_a]

Try the Pierce Western Handbook and Troubleshooting Guide
The Tech Tips & the Application Notes.

[psingh]

[Only registered users see links. ]

Look at "Troubleshooting guide" section. It covers Everything out there.

[lovelyday713]

I use any type of dryer sheets like the ones you put in the dryer to remove static from your clothes! They have many uses!

[Allison M]

Originally Posted by lovelyday713

sorry..

3) Static signals on the film
These look like blobs everwhere. sometimes caused by static, to get a really nice looking blot, I take my probbed nitrocellulose, after putting ECL and place it in a plastic sheet protector. I then reduce the static by wiping down with a dryer sheet. And then finally put the film on to develop... removes static everytime!

I dot the reagent on a glass plate (from the Ruby apparatus). Then place the membrane(s) face down in it. I've never had problems with static and background is minimal.

[bwbrian]

When you've done some optimization, but the results are still not clean on film, Pierce sells a solution (called Erase-It) that can be used to remove background from the film - even after it's been in your lab notebook for awhile.

[meenurohini]

Originally Posted by danfive

I also recommend using the Storm Scanner (GE) for image capture/blot analysis.

i have been doin western blots for few months nw...since last month i started this problem of fading of the bands...the bands fade too quickly and so i cannot take the second or longer exposure...i havent changed anything in the protocol...i read that substrate depletion is possble cause for this which can be due to too much of HRP...but i havent changed the secondary concentration....can there be any other reason ??????

[danfive]

Originally Posted by meenurohini

i have been doin western blots for few months nw...since last month i started this problem of fading of the bands...the bands fade too quickly and so i cannot take the second or longer exposure...i havent changed anything in the protocol...i read that substrate depletion is possble cause for this which can be due to too much of HRP...but i havent changed the secondary concentration....can there be any other reason ??????

So you have luminescence, and it fades way too quickly.

That means you have too little of ECL reagent (could diminish with time), or too much HRP.

Too much HRP could be due to too much target protein, which then binds too much primary antibody, then too much secondary-HRP conjugate. (Since the protocol hasn't changed: probably don't have too much 1st or 2nd antibody, though dilutions or concentration would be helpful).

try reducing the amount of sample loaded (concentrations would be useful in troubleshooting).

[meenurohini]

thanks...wd be doin that...