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What is the cause of this Vertical smear in my western blot?


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#1 jcsslug

jcsslug

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Posted 22 July 2014 - 02:33 PM

HI

   I was wondering if anyone had an idea as to what is going on with my western blot. The issue I have is that when I develop my blot for P-AKT or Total AKT I get vertical smears in every lane when I do long exposures. The strange thing is that the vertical smears do not appear when I probe for proteins with lower molecular weights ex P-ERK, Total ERK Sprouty, GAPDH, Adiponectin. When i do long exposure the bands are blown out but there are no vertical smears appearing. I don't think its sample degredation, becuase I would assume that would affect all proteins uniformally, and I would see smears in the P-ERK, Total erk etc. The coomasie stain is not the greatest. There were bubble etc. but i can see clear protien bands and it looks nothing like images i've seen online of western blots with degraded samples.  I have not probed anything with a higher molecular weight (we're not interested in anything larger than AKT 60 kd, for now) so I don't know if this affects all proteins 60kd or higher.

    I first thought it could be an issue with the cell lysis buffer.

      Recipie: 1x Cell lysis buffer:

         1 ml 1x CLB

          1 ml 10x phosphostop

         80 ul of 115 mM PMSF

         7.92 ml Miliq H20

 

  I made fresh CLB and then ran the gel again, and encountered the same problem. This time I've made fresh CLB and Fresh Loading Buffer. The loading buffer has DTT and BME in it. I definitely left the loading buffer out for a couple of hours previously. The samples are boiled for 5 min at 95C then put immeidately on ice. After they have cooled I vortex them quickly and then spin them quickly to bring down the condensation on the lid. THe running buffer and transfer buffer are made fresh everytime. My lab uses the NuPage 20x concentrated running buffer and transfer buffer sol'n. The gel is run at 90 mv for 150 minutes and the transfer occurs for 1 hr at room temp at 30mv. I'm loading my lanes with 5 ug of either mouse liver lysate or mouse muscle lysate. Its not an issue of running the gel or transfer step. I've been running these gels for months and that hasn't been an issue. I don't think its an anitbody issue. I've been using a 1:4000 dilution for my primary Cell signalling Rabbit- Anti P-AKT or Total- AKT. I thought the vertical smearing may be due to too much AB but previous gels have used the same AB concentrations and have worked well, so it can't be that.

 

 Images below:

    The third image Total ERk looks fine even though some of the samples are blown out. The first two images (AKT) on the other hand have the vertical smears I was talking about. I cropped the image but they extend the entire length of the blot.

  Thank you!!!

Attached Thumbnails

  • COOMASIE_STAIN_IMAGE_WESTERN_BLOT_071514.jpg
  • 1_min_akt.jpg
  • 10_min_p-akt.jpg
  • 10_min_p-erk.jpg

Edited by jcsslug, 22 July 2014 - 02:34 PM.





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