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
- - - - -

Strange bands and large smear in western blot


  • Please log in to reply
4 replies to this topic

#1 nanobio

nanobio

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 20 July 2011 - 11:21 AM

Hello everyone,

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

Attached Files

  • Attached File  blot.bmp   79.68KB   246 downloads


#2 casandra

casandra

    carpe diem by the jugulum

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,034 posts
56
Excellent

Posted 20 July 2011 - 11:44 AM

Hello everyone,

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

These three are identical samples? What have you done (or changed) since you started getting these weird results?
"Oh what a beauteousness!"
- hobglobin, personal comment about my beauteous photo......

#3 nanobio

nanobio

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 20 July 2011 - 12:17 PM

Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.





Hello everyone,

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

These three are identical samples? What have you done (or changed) since you started getting these weird results?



#4 casandra

casandra

    carpe diem by the jugulum

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,034 posts
56
Excellent

Posted 20 July 2011 - 12:37 PM

Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.

have you tried preparing fresh reagents esp the antibodies? Anyone else in your lab having the same inconsistent results? You've to do a process of elimination here...
"Oh what a beauteousness!"
- hobglobin, personal comment about my beauteous photo......

#5 nanobio

nanobio

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 20 July 2011 - 01:28 PM

Yes, i prepared fresh reagents and antibodies, Unfortunately nobody else works with these Abs in lab.
Gonna try and do that casandra, eliminate step by step and try to find the problem.


Thans for your help.



Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.

have you tried preparing fresh reagents esp the antibodies? Anyone else in your lab having the same inconsistent results? You've to do a process of elimination here...






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

©1999-2013 Protocol Online, All rights reserved.