Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

"Sandy WB"


  • Please log in to reply
9 replies to this topic

#1 Fly78

Fly78

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 18 October 2010 - 09:10 AM

I have been spending my life recently trying to make my WBs work again...
They look "grany"...not sure how to explain but basically, very dirty, full of non specific bands and with a "sandy" texture...

I am trying to detect pAkt (and also pS6K) and I cannot get a decent signal.
I've been following a protocol given to me by my boss, and apparently it seemed to work for him.
I prepared all my solutions (Tris buffers for the gel, Transfer buffer...) from scratch more than once but no result.
I used a different secondary to see whether the problem could come from there, but the result was the same.
I blocked in 5%milk-PBST or 5%BSA-PBST, and again nothing.
I tried to cast my gel fresh on the same day I am running them (and not the day beofre, storing at 4C as I was doing before), nothing...
I am really desperate to get these WB to work because they're really crucial for my PhD thesis and I don't have that much time.

I am thinking about other possible options:
-using TBS rather than PBS
-changing the membrane (borrowing some other nitrocellulose membrane, mine could have gone bad misteriously)

Can anyone please give me any suggestion?

Thanks a lot

#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 18 October 2010 - 11:56 AM

How are you doing the transfer? I've seen things similar to what you describe when using the X-Cell blot module (from Invitrogen) and you forget to use Whatman paper in your transfer stack such that the gel and the membrane are in direct contact with the sponges -- this results in a "pebbly" or perhaps "sandy" look...

#3 Fly78

Fly78

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 18 October 2010 - 12:08 PM

How are you doing the transfer? I've seen things similar to what you describe when using the X-Cell blot module (from Invitrogen) and you forget to use Whatman paper in your transfer stack such that the gel and the membrane are in direct contact with the sponges -- this results in a "pebbly" or perhaps "sandy" look...


I am using the Trans-Blot Semi-dry (BIORAD) and I use three layers of filter paper (pre-wet with transfer buffer)on each side...

#4 ksturm

ksturm

    member

  • Active Members
  • Pip
  • 13 posts
1
Neutral

Posted 19 October 2010 - 05:16 AM

just a suggestion: clean your sponges carefully. With mild acid or sth like that. After longe usage they can be soiled by proteins.

#5 Fly78

Fly78

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 19 October 2010 - 06:08 AM

I am not actually using sponges, as I said, I am doing a semi-dry transfer and use 3 layers of filter paper on each side...

#6 ksturm

ksturm

    member

  • Active Members
  • Pip
  • 13 posts
1
Neutral

Posted 19 October 2010 - 08:09 AM

Sorry, my fault. Biorad semidry is actually very good system. 'sandy' appearance dues to problems during transfer. Beside nitrocellulose pvdf-membranes are recommended for the biorad System. But 10-15% of protein pass pvdf-membrane without binding. What is your transfer Buffer? Unspecific bands are due to blocking or primary ab. Some people add Tween20 to their blocking solution.

#7 elaila

elaila

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 12 January 2011 - 12:47 PM

I've had this problem before when trying to blot for a membrane protein. I blocked membrane in 5% milk overnight to get rid of the grainy appearance on film. Hope this helps.

#8 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,240 posts
336
Excellent

Posted 12 January 2011 - 03:04 PM

What developing system are you using (HRP, alkaline phosphatase?), what sort of film?, how old are the film processor chemicals?

#9 steffi333

steffi333

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 13 January 2011 - 06:00 AM

I have been spending my life recently trying to make my WBs work again...
They look "grany"...not sure how to explain but basically, very dirty, full of non specific bands and with a "sandy" texture...

I am trying to detect pAkt (and also pS6K) and I cannot get a decent signal.
I've been following a protocol given to me by my boss, and apparently it seemed to work for him.
I prepared all my solutions (Tris buffers for the gel, Transfer buffer...) from scratch more than once but no result.
I used a different secondary to see whether the problem could come from there, but the result was the same.
I blocked in 5%milk-PBST or 5%BSA-PBST, and again nothing.
I tried to cast my gel fresh on the same day I am running them (and not the day beofre, storing at 4C as I was doing before), nothing...
I am really desperate to get these WB to work because they're really crucial for my PhD thesis and I don't have that much time.

I am thinking about other possible options:
-using TBS rather than PBS
-changing the membrane (borrowing some other nitrocellulose membrane, mine could have gone bad misteriously)

Can anyone please give me any suggestion?

Thanks a lot



In the past I've run gels where the glass plate over the front has come loose. Sometimes this causes protein to leak out of the front of the gel and it can make the blot look a bit grainy.

Also, to maximise your blocking, try diluting antibodies in blocking buffer rather than just PBS - should help to minimise background. And (you probably do this already) make sure you wash in PBS with Tween in it and wash at least three times with high volumes of wash buffer.

As for non-specific bands, are you lysing your cells in buffer with a protease inhibitor? Sometimes breakdown can occur during lysis. I've also noticed I get some non-specific bands around the same weight as my protein of interest if the cells are too highly confluent when I lyse them.

#10 Say Chi Sin Lo

Say Chi Sin Lo

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 20 January 2011 - 02:53 PM

I am not actually using sponges, as I said, I am doing a semi-dry transfer and use 3 layers of filter paper on each side...


Try using 6layers of filter paper on each side. So 12 layers per gel.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.