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Cell Lysate Western Blot Little or No Signal


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#1 alphawhiskey

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Posted 15 January 2014 - 09:18 AM

Hi all,

 

I have Cos-7 cell lysates that I ran a western with. I loaded around 60 ug total protein to each lane, along with the purified version of the protein I am detecting. I was able to detect the purified protein down to 50-100 ng, but I am not getting any signal from the cell lysate. I then blotted for actin, and saw a very faint signal from the cell lysates. Does this mean I am not loading enough protein? How can I concentrate the proteins in my lysate?

 

Thanks!



#2 Missle

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Posted 16 January 2014 - 04:57 AM

Without knowing what the protein is that you're trying to detect or the sensitivity of your western....60ug total protein seems like plenty to me.  Purified protein detection down to 50-100ng is okay sensitivity but not stellar.  Do you have an idea of the level of expression of the protein of interest in your lysate?  Are there protease inhibitors in you lysate?



#3 alphawhiskey

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Posted 16 January 2014 - 09:09 AM

We do not have much idea of the level of expression of the protein is. I put 1mM benzamidine HCl in the cell lysis buffer.



#4 jerryshelly1

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Posted 16 January 2014 - 09:33 AM

60ug is quite a bit a protein and at that concentration you should have no trouble getting a very robust actin band.  If you are getting a decent signal from your positive control on the same blot, you can probably rule out everything post protein denaturing (check your blot via poncea-S and commassie stain your gel post transfer, just to make sure something funky didn't happen - very unlikely though).

 

If I were you, I would harvest more cells and do a milder protein extraction. Is this your first time working with these cells?



#5 alphawhiskey

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Posted 16 January 2014 - 05:15 PM

With Ponceau S, I can see bands in the cell lysate lanes, which should mean there is protein. My lysis buffer only had 0.05% Triton X; I used sonication to lyse the cells (3 pulses of 3 seconds, with 25 sec intervals). Could sonication have harmed the proteins? 

 

This is my first time working with Cos-7 cells, and I am quite new to western blotting. 



#6 jerryshelly1

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Posted 16 January 2014 - 05:26 PM

It is definitely a possibility. That particular protein may be more susceptible to sonication than other proteins. A quick check would be to prepare your purified protein in the same manner (even though it is already purified). You could potentially take 100ng of your purified protein and add the lysis buffer and sonicate. Run your samples on a dot blot and see if this destroyed the antibody recognition site. I only suggest doing a dot blot because they can be completed within 3hrs as compared to a two day western blot (depending on your protocol).

 

Have you tried just adding lysis buffer to your Cos-7 cells, mildly vortexing, and incubating on ice for 30 minutes, spinning down and removing the supernatant? This is significantly milder than sonication and may or may not solve your problem.

 

If your protocol was given to you and sonication has worked in the past, maybe you should double check your output settings.

 

I haven't worked with Cos-7 cells, but I do work with HEK cells quite frequently.

 

Edit - Is this protein expressed from a plasmid that you transfected? If it is, maybe you should take a step back and see if you have mRNA expression via a quick PCR reaction. You don't make it clear as to whether you purified the protein or if the protein is a manufacturer recombinant protein.



#7 alphawhiskey

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Posted 16 January 2014 - 06:06 PM

Thank you for your advice! I can definitely try treating the purified protein the same way and see what happens. 

 

When I was lysing the cells, I had incubated them in the lysis buffer on ice for about 20 minutes, but when I checked them under the microscope, there were still whole cells floating around, so I thought they didn't completely lyse, which was why I sonicated them. 

 

Perhaps if I use 1% Triton X instead, then the cells would completely lyse on ice? 

 

We are just trying to detect the endogenous protein; no treatments were done to the cells prior to lysing. The protein was purified by the lab, but the antibodies were purchased from Santa Cruz. 



#8 jerryshelly1

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Posted 17 January 2014 - 07:46 AM

That may work. If you didn't know... This website (http://highwire.stanford.edu/) is a really good reference when you like to search by key words. Maybe try searching for your protein + cos7 + lysis to see what pops up.






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