emou

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!   i will do it again again to test what is going wrong....

Thank you!!!

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!!!!!

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!

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!!

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!