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western blot of cardiomyocytes


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#1 helencollins560

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Posted 16 August 2013 - 02:37 PM

Hi there

 

Im new to western blotting

 

I have recently tested a new method of isolating cardiomyocytes and extracted protein.  Ran a gradient gel (4-15%) of my samples at 10ug protein.

I notice on ponceau staining that it is weak, probably since ive loaded 10ug.  I cut the blots as my protein of interest is 80kda and my control gene GAPDH is ~37kDa.  I notice on probing for GAPDH that it is slightly larger than 37KDa (its more like 40-45kda). It is extremely hard to probe for my protein of interest as it is weak in these samples

 

Just wondered whether there was a chance that the protein is degraded some how? If so, would i still be able to use these samples to look at my protein of interest.  Or would i just need to load more protein than 10ug

 

any help is appreciated as im very much a novice at this technique

 

collins560



#2 loz

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Posted 18 August 2013 - 01:29 AM

Hi!

 

Not sure that this will help. I am not a blot master and ppl think differently about these things but:

 

Never worry about Ponceau, in fact I don't bother with it. If you do the ponceau and don't see anything, run your blot anyway....far more sensitive. 10ug should be more than plenty depending on your protein and the sensitivity of your antibody. I'd think from what I have worked on that this is on the excessive side. My second point is that molecular standards vs. sample are never exact, not like doing a DNA agarose gel at all, it is very round about. It might be an ok size. Cant you also have another control for product size of your protein of interest along side this as well as GAPDH which I assume you are using to see if the blot/antibody has worked? (question why is GAPPH your sole control?) Lastly, degraded proteins you will know, your blot will look...."blotchy", unless you have over loaded it with too much protein, that will look kind of the the same. Did you do a coomassie stain or other? How did that look? Why don't you post some pics of your coomassie or what ever and your blot on this thread along with some labels/explanation of what we are looking at and your ladder (and what kind it is)? It might help ppl with your question.

 

Just a last little thing. If you are storing proteins and are worried about degradation....I really don't like freezing them (not to say that  you cant)....but put a little sodium azide in there to keep it and some PMSF to inhibit proteases is a really good thing.

 

Cheers!!!!!!



#3 rkay447

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Posted 19 August 2013 - 09:37 AM

Well, depending on the abundance of your protein and the affinity of your antibody, 10ug may not be enough.  You say that the protein is "weak" and I assume it is expressed at a low level.  10ug is a very small amount of protein for blots.  Typically I start at 40ug.  Also, there are rare situations when ponceau inhibits or blocks antibodies.  This is a rare event but... who knows.  I never ponceau until after I'm done with all antibodies.  The size a protein appears on a gel can vary quite a bit depending on the percentage of gel so your gradient gel might not give you quite the proper size for GAPDH but if you are getting one discreet band around the correct size, I'd say it's good.  There is a possibility that your protein of interest has degraded but unlikely if you used protease inhibitors and kept your sample cold during harvesting.  I never had issues with degradation from cell culture.  Yeast, however, is an entirely different beast.  That being said, some proteins are more sensitive and unstable than others.  You might just be working with an unstable protein.  There are some options to help you if that is the case but for now, if I were you, I'd run a gel with more protein.  I am willing to bet that you didn't run enough sample to see your protein.  Do you have a positive control for the western blot so you know your antibody is good?  Have you tried overnight incubation with a strong dilution?  What ECL substrate are you using for detection?  Piece puts out a femto that is very sensitive.  






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