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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Western Blot Background Troubleshooting

Western Blot Background Troubleshooting - Western Blot Forum

Western Blot Background Troubleshooting - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #11  
Old 06-08-2008, 08:36 PM
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Talking Re: Western Blot Background Troubleshooting

My tip is:

Use a leveler to make sure the proteins are at same level.
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  #12  
Old 07-01-2008, 09:24 PM
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Default Re: Western Blot Background Troubleshooting

Try the Pierce Western Handbook and Troubleshooting Guide
The Tech Tips & the Application Notes.
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  #13  
Old 08-29-2008, 09:04 PM
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Default Re: Western Blot Background Troubleshooting

[Only registered users see links. ]

Look at "Troubleshooting guide" section. It covers Everything out there.
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  #14  
Old 01-14-2009, 07:51 PM
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Default Re: Western Blot Background Troubleshooting

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!
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  #15  
Old 01-15-2009, 03:44 PM
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Default Re: Western Blot Background Troubleshooting

Quote:
Originally Posted by lovelyday713 View Post
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.
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  #16  
Old 04-03-2009, 06:05 PM
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Default Re: Western Blot Background Troubleshooting

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.
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  #17  
Old 10-06-2010, 12:30 PM
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Default Re: Western Blot Background Troubleshooting

Quote:
Originally Posted by danfive View Post
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 ??????
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  #18  
Old 10-06-2010, 04:34 PM
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Smile Re: Western Blot Background Troubleshooting

Quote:
Originally Posted by meenurohini View Post
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).
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  #19  
Old 10-07-2010, 11:49 AM
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Default Re: Western Blot Background Troubleshooting

thanks...wd be doin that...
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