Dear Samrina,
A couple of questions to help me to think:
The target protein is supposed to be cytosolical or exposed to the extracellular domain? Did you permeabilize your cells before the incubation with the sera? Did you perform a blocking step before the incubation with the human sera (I mean with purified human albumin for instance)? Which is, exactly, your secondary antibody? and If you add the secondary antibody without adding the sera did you still observe the false positive signal?
Cheers,
Dear Sebastian,
The target protein is in caveolae .I do not perform permbealisation step as my protein is in caveolae.I have tried blocking with 2-5%BSA ,10%goat serum as my secondary is raised in goat.lI do not get false positive signal in my only secondary antibody coverslips.
Regards
Samrina
Dear Samrina,
Since your secondary antibody alone did not produce the false positive, I would tend to think that "sticky" components on the human seras (both in control and patient) become adsorbed on the cells and also interact unspecifically with the secondary antibody. Moreover, this signal most likely is masking the real signal you are expecting to detect. That's why I would recommend to try different blocking agents to try to cover those epitopes recognized by the sera components. You can try human albumin, protein free blocking agents (Pierce), and if your protocolo allows it add some tween to the blocking agents.
What did you use to dilute the sera fractions?
Best,
Dear Sebastian,
Thank you so much for your kind suggestions.I will try using human albumin as blocking agent .
I have tried two methods of immunoflouroscence given below:
1.In first case if blocking is done using 2-5%BSA or 10%goat serum for an hour at room temperature.In this case I dilute my antibodies in 1%BSA or goat serum.I
2.In other method I have tried omitting the blocking step .So,in this particular method I dilute my primary and secondary antibody in PBST(TWEEN 20)0.2%.
Additionally ,as I have mentioned above that I use polylysine to adhere my cells ,could this be one of the reason to give false positive signals?.
Regards
Samrina
.
Samrina,
I'm not ruling out your hypothesis that Polylysine may have something to do with what you see, but it is highly unlikely. Besides, If that was the case I would expect to see much higher unspecific signal as background meaning "surrounding" the cells instead of "on" the cells.
Perhaps another possibility to check is to perform a blocking step after the addition of the sera. This could hep to hide those sticky epitopes from the sera (that then adhere to the secondary antibody) but still allow recognition of specific antigens by the antibodies in the patient's sera.
Protocol 1 (2 blocking steps): Fix>wash>block>sera>wash>block>secondary>wash
Protocol 2(1 blocking step): Fix>wash>sera>wash>block>secondary>wash
Just to clarify my comment, you will be blocking the first time to hide cell's unspecific epitopes from the antibodies in the sera, and a second time to hide sera's unspecific epitopes from the secondary antibody.
Hope it helps,
Dear Sebastian,
Thank you for the suggestions.I will try the aforementioned protocols.
Regards
Samrina
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