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Low signal, weird background stains on blot


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#1 alphawhiskey

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Posted 04 February 2014 - 02:10 PM

Hi all,

 

I am trying to detect a ~80kDa protein in HEK and Cos cell lysates. I use the purified GST-tagged protein as control and standards on the western blot. Last week, when I used 5 - 50ng of the purified protein for the controls, I got very clear bands. But this week when I ran the western blot again using the same proteins, I got very low signal. I am also getting some weird stains on the blot. 

 

For the primary Ab, I use 1:200 and incubate overnight at 4 degree. For the HRP-conjugated secondary Ab, I use 1:6000 and incubate for 1 hour at RT. I rinse 4x 5-7min after each incubation, and use Pierce ECL kit and the LICOR system to visualize the blot. 

 

I have attached one western blot from last week when it was fine, and one from this week where I could barely get any signal. 

 

What could be causing this issue?

 

 

Thanks!

Attached Thumbnails

  • Bethyl rabbit Ab.jpg
  • Bethyl rabbit Ab worked.jpg


#2 bob1

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Posted 04 February 2014 - 04:47 PM

I would say you have a lot of background staining in both blots, which either indicates that you haven't blocked the membranes enough, or that your secondary is too concentrated and/or incubated for too long.

 

However, the swirly background you see is due to liquid swirling between the membrane and gel during transfer.  Light bands on a dark background can indicate that there is too much protein present in that lane (essentially burnout of the HRP).  Spots on the membrane can come from a number of sources, dirt, forcep marks, precipitated antibody, overlapping membranes, and nicks/holes and folds in the membrane can all cause these.

 

The reason you can't see your bands this time around could be a huge range of things - is the protein stable? Are the antibodies stable?  Did it transfer properly (ponceau stain?)? Did you make your PBS-T/TBS-T properly?  Did the membranes dry out...



#3 alphawhiskey

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Posted 05 February 2014 - 10:24 AM

Perhaps the protein is not stable. I do not know how stable the antibody is; I only recently purchased it. Is there a way to find out? 

 

I used the same transfer procedure for both blots, and the same batch of TBS-T.

 

I don't think the membrane dried out, but would it not work if the membrane dried out at some point?



#4 bob1

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Posted 05 February 2014 - 01:01 PM

The datasheet is the best place to start for antibody stability - it may well say storage conditions.  I know of a few primary antibodies that will only last a matter of a day or two in the fridge, whereas others may last years.  You could strip the blot that worked and re-probe with the antibody to see if it is an antibody problem

 

Drying of the blot is not a problem usually, but can cause high background especially if dried before blocking.



#5 Missle

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Posted 06 February 2014 - 05:01 AM

Is it nitrocellulose or PVDF?



#6 alphawhiskey

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Posted 10 February 2014 - 11:53 AM

It is nitrocellulose.

 

I repeated the blot, used new stock of purified protein and it worked. But I am still getting high background.






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