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Western blot and immunofluorescence staining


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#1 E-biologist

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Posted 20 February 2013 - 12:25 PM

Hello everyone!!

Could someone please explain to me what are the possible reasons when immunofluorescence staining is positive while the western blotting is negative?
The antibody i used in both techniques is the same.
For western blotting, I loaded 6 samples in the membrane and i had positive staining for my antibody in 5 of them. I repeated the experiment twice and i had the same results. But to my suprise, when i carried out immunofluorescence, i had positive staining in all of my samples and of course this was a positive and not an unspecific staining.
Also, when i used in the same membrane another antibody, this worked very good for all my samples.


Thank you in advance!!!

#2 jerryshelly1

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Posted 20 February 2013 - 03:26 PM

Your post is a tad confusing. So I am assuming you are using a fluorescent tag? Or are you doing immunohistochemistry? Either way if you are seeing a clear fluorescence in your samples, but not in your prepared protein samples (Western blot); I would say you messed up the protein isolation for your 6th sample. Re-isolate protein from that cell and see what happens.

Good luck

#3 E-biologist

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Posted 20 February 2013 - 09:50 PM

Thank you for your advice!
I am doing cell fluorescence immunostaining and not immunohistochemistry.
I isolated protein from these cells twice...but to be more accurate...i isolated protein twice and the concetration was quantified using Bradford assay and i had protein!!!
I confirmed my trasfer using ponseau for my membrane and commassie blue for my gel and i had no proteing in gel but i had in my membrane. I used GAPDH to test my equal loading and everything was ok!!!
I suppose that if i had problem with my protein extraction, i wouldn't see GAPDH staining... or not??

I'm so confused!!it's the first time that something like this happens to me and i don't know what to do!

#4 jerryshelly1

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Posted 21 February 2013 - 02:25 PM

Bradford only looks at total protein concentration, it may be possible that your protein isn't in your sample or is lost in some way (epitope recognition gets altered?).

Your protein may possibly be degraded under your standard protein extraction conditions. GAPDH is fairly stable during protein isolation. Is your protein known to be modified in anyway (ubiquitin, etc. - degradation during isolation)? Do you have an overexpressing vector containing your protein of interest (use this to increase protein concentration to verify its presence)? Are you using similiar Ab's between staining and WB? Are you sure that your protein is localized in the cytoplasm? Is it possibly that your protein extraction does not fully rupture the nucleus where your protein resides?

Are you 100% sure the staining you are seeing is your protein? Does your staining control look correct?

May seem like stupid questions, but maybe that will get your thoughts rolling in the right direction.

Good luck.

#5 bob1

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Posted 21 February 2013 - 04:25 PM

Could be that the antibody works better with native protein than denatured, could be that there is more of the specific protein in some samples than others (i.e. detection limit).

It could be that in your immuno you are actually seeing a cross-reactive protein in all cases, as well as specific staining (I would think this most likely), test by blocking the staining with peptide for the epitope of your antibody.

#6 E-biologist

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Posted 21 February 2013 - 09:40 PM

Really Thank both of you for your time!!

I just understood that i deleted some information by mistake..

The 5 samples in my western blot were cells that i isolated from a tissue and the 6th sample were the same cells that i purchased from a company (primary cells, not a cell line).The antibody i used was the marker of these cells. So, if they sell to me these cells, i had to see my marker protein in these.. I sent an email in the company and they told me to increase the protein amount for the western blot and the exposure time. I did both of them, i exposed the film for 2,3 & 4 hours but still there was no signal of the marker in the 6th sample.
It's the first time i work with cells that i purchased from a company.

They say in the datasheet that the cell stain positive in this protein by immunofluorescent but it is logical that i cant see this in western blot?I use the same Ab for western and cell immunostaining. I used it again in the past when i wanted to confirm the purity of the cells that i isolated and it worked very well. I am 100% sure that the staining i saw was my protein. Positive and negative control worked really very good. Also my protein is not modified during isolation. They are not stupid questions..they help me! Posted Image

What i can not understand is that the Ab in the western blot works very well for the cells that i isolated and not for these that i purchased??

I will try what you recommended for the situation, a vector, a peptide and i hope to see something!!!

Thank you both of them again!! Posted Image Posted Image Posted Image

Edited by emou, 21 February 2013 - 09:43 PM.


#7 jerryshelly1

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Posted 22 February 2013 - 06:46 AM

Stupid question, but if the protein is a cell growth marker are you 100% sure that the cells are at that point in there cell cycle. Do they show any indicative morphological characteristics? Can yo double check with another marker that is expressed during the same period?

#8 E-biologist

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Posted 23 February 2013 - 11:48 PM

When i cultured them they showed all the morhological characteristics for these cells...
My purpose was to check 2 markers for these cells and i will do it tomorrow...
but that was the most important marker for these cells and they had to stain positive in this....i can not undestand why this happens...positive in cell immunostaining and negative in western blot....really i'm confused.....
I'll see what will happen with the other marker tomorrow....

Thanks you again for your advice and your time!!!!!

#9 jerryshelly1

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Posted 24 February 2013 - 09:40 AM

So weird. Have you ponceau stained your blot to make sure you have efficient transfer of your protein? Coomassie to make sure there is protein on your blot?

#10 E-biologist

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Posted 24 February 2013 - 11:23 AM

ohh yes!!!i did it!!! it was just a wonderful western blot! hahah!!
i don't know and i can not understand what happens....
i will repeat the western for the thirth time and i will prove my mebrane and with the second marker....if it will not work again i dont know that to do...
but as a collegue of my lab said...search and research!!that why we are researchers! Posted Image i will do it again again to test what is going wrong....

Thank you!!! :)




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