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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


serum sample.....

serum sample..... - Western Blot Forum

serum sample..... - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 09-10-2010, 10:59 AM
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Default serum sample.....



hi...
I'm new...and my english isn't so good...so please forgive me!!
I've some trouble with western blot.
I'm trying to identify 2 serum protein and in my gels I load the whole serum.
the principle problem is that I've to thin down samples but I don't know in which buffer. at present I 'm thining down my samples in water and then I put the sample buffer...

somebody can give me a suggestion?
because the separation is bad...some wells seem to be more narrow than others....
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File Type: jpg Sara gel comassie 12.08.jpg (73.0 KB, 20 views)
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Old 09-17-2010, 10:13 PM
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Default Re: serum sample.....

I don't understand why you need to thin down samples. I've worked with serum samples before and we always used them as is. Although the water should not pose a problem, you could add equal volume of sample buffer to serum samples only.

Are you using a protein assay to check the protein concentration of the samples?

It seems like unequal loading of samples is the problem in the western blot you have attached. This means you have more micrograms of proteins in some lanes than you do in some of the other lanes. This is controlled for by using a protein assay to measure the protein concentrations of different serum samples.

Do you have a loading control protein? It may be that the concentration of the protein varies across different samples.

When you say the separation is bad? Do you mean vertically or horizontally?
Also, you expect the more lanes you have in a gel the less separation you will give horizontally. If you are talking about vertically then you can run the gel longer or use a higher percentage gel.

Last edited by MJD; 09-17-2010 at 10:19 PM.
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  #3  
Old 09-20-2010, 02:26 PM
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Default Re: serum sample.....

thank you...
i need to thin down my samples because they are very concentrated and I want to load only 20ug of total proteins. I solved this problem thining down with running buffer.
before loading i check my concentration with bradford assay. but... may be...i could fault to check it.
the other problem is that I've not a loading control and this makes complicated analisys of my results...
i'm trying to solve all this problems...
if you have any others suggestions....

thanks

Sara
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Old 09-20-2010, 04:27 PM
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Default Re: serum sample.....

Your pic looks fine, I did notice some difference in the lane widths. But that is probably due to running ~17 lanes. That's a lot of samples for this one gel, and that is likely what causing the slight distortions---overall your coomassie stained gel is certainly of acceptable quality.

I wouldn't dilute with water, just a waste, put your serum in sample buffer or Laemmli Buffer.
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Old 09-24-2010, 11:27 PM
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Default Re: serum sample.....

Hi Sara,

This definitely looks to me like overloading of some wells with protein. I agree with MJD - you should check the amount of total protein by Bradford or BCA, and load the same amount of total protein in each lane. That should give you at least one band across the gel (or western) that is the same in every lane, depending on what your samples are it might be B-actin or something else. Repeat your protein assay and make sure that all of your measurements are within the linear range to be sure that the total protein measurement is accurate. Good luck!
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Old 09-28-2010, 08:31 AM
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Default Re: serum sample.....

I solve my problems! I check again the concentration and I load the same amount in each lane...
now..I've another problem...
my protein of interest is a tetramer so I've thinked to make a NATIVE PAGE...but somenthing was wrong,in western blot I don't see the band at the aspected mw, but the same of SDS PAGE, the monomeric form.
I've used biorad precast gel that, they say,are suitable for native page and I've prepared my samples without 2-mercapto ethanol.
i don't know what's happened, somebody can help me?
it's strange because the same protein in the monomeric form and oligomeric form are both present in my DIGE gels..
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Old 10-08-2010, 03:52 PM
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Post Re: serum sample.....

Quote:
Originally Posted by sara.bi View Post
I solve my problems! I check again the concentration and I load the same amount in each lane...
now..I've another problem...
my protein of interest is a tetramer so I've thinked to make a NATIVE PAGE...but somenthing was wrong,in western blot I don't see the band at the aspected mw, but the same of SDS PAGE, the monomeric form.
I've used biorad precast gel that, they say,are suitable for native page and I've prepared my samples without 2-mercapto ethanol.
i don't know what's happened, somebody can help me?
it's strange because the same protein in the monomeric form and oligomeric form are both present in my DIGE gels..
You can take a lot of precautions with protein preparation. But it is a fact that a single protein will exist in various forms. So it is not strange that you have some monomeric and dimeric forms. You probably have soluble and insoluble forms (just can't see it), or different states of phosphorylation, glycosilation etc that show up at different pIs.

You can only avoid this by deliberately picking proteins that are so well characterized in the literature, that you can say it will show up in a single native state band. Or chemically treat to reduce, dephosphorylate etc before running them.
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  #8  
Old 10-13-2010, 01:11 PM
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Default Re: serum sample.....

thanks...but my problem is different...
I know that my protein has different form, it has a tetrameric form in a native status..but also monomeric.
what I would like to do is to run my serum samples and observe this native form; but all my test were wrong. I've tried with a gradient gel, a 12% gel and 6% gel...but nothing...
any suggestions?
thank you so much
Sara
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