blotchy, irregular bands

[rufus]

I have run several blots but all my bands turn up irregular, never uniform-
The bands are hazy and either side of the well is sometimes more full than the other. I sometimes get these diamond shaped images where the protein of interest should be...it is like its overpowering my labeled bands.
No problem with non-specific binding, but the protein of interest is running lower (smaller) than it should. I use 1xPBS w/.1%tween in my 5% milk blocking solution and use the 1xPBS w/.1%tween to incubate my primary and secondary in. I always stain my gel to make sure the transfer was good so I don't think the transfer is the problem.

suggestions on how to troubleshoot and crisp up the blot would be greatly appreciated!

pic included,
Thanks

[jiajia1987]

"I use 1xPBS w/.1%tween in my 5% milk blocking solution and use the 1xPBS w/.1%tween to incubate my primary and secondary in. "

Try incubating your primary and secondary antibodies with your blocking solution instead and use the 1xPBS w/.1%tween for washing. Dilute your samples too and load them ont he gel to run again. Hopefully.. this works..

[Anastasia_S]

do you use photoshop to clean images? It helps to enhance/decrease colour and sort of clean up the image (generally using "curves"), and because you do it to the whole image, not parts of it - it is considered ok

[admin]

Hello there, looking at your gel I think you have loading issues and possibly gel or even transfer issues.

Are you loosing any sample while loading (is it puffing out of the well?) - you could try increasing your sucrose.

How does your ladder look - are the bands quite sharp and well balanced in darkness? You may need to mix your SDS-PAGE gel a bit better and use a stacking gel as I see your bands are a bit unformed.

During transfer, you may have small bubbles i see missing spots in your bands. Try to get rid of bubbles with a roller or broken plastic pipette.

Let us know how your progress goes please post your next gel image / blot image.

cheers

[Tfal]

Photoshop your blots... that's a slippery slope! Increasing contrast (reducing uniform background) is acceptable, but retouching your bands: dubious! Many publications now ask for your entire and unprocessed blots

[Tfal]

ya anyway rufus, you might wanna play with your voltage (probably lowering it) during electrophoresis and/or transfer.
About "the protein of interest is running lower (smaller) than it should" Well it might not be your protein! (non specific binding of 1st or 2nd AB due to degradation or unsufficient blocking) Or it might have been lysed (unsufficient protease inhibitors, too much free/defreeze and/or not kept cool enough)!

good luck

[piramses]

do you have a positive control? also run it too and if you have the same problem then i think this is due to your gel or the apparatus. well, i had an experience with this, there was bubbles forming under the gel, i would stop the run and remove the bubbles carefully with a needle-and continue. also i would recommend you not to run your gel fast-i do at max. at 90V.
looking forward for your progress

[Tiffy]

Hello everybody,

I am so happy that Piramses has posted this problem, because I have the same problem. Could it be that it is a problem of high molecular weight proteins. I have the impression that I get this result with big proteins.
Could it be that my sample buffer (2x Laemmli) is not sufficient for my protein amount?
Shall I soak my gel in the transfer buffer after running?
Any comments?
Thanks a lot
Tiffy