Hello!
I tried to block my fixed cells on 10% FBS and 5% NGS (Normal Goat Serum) in PBS1X for 1h at RT and then incubate with a primary Ab anti-Mouse (4 °C ON in 10% of the blocking solution) and a secondary Ab anti-Mouse raised in Goat (2h RT in 10% of the blocking solution). The result was awful, so much background and everything was like stained. Then I totally changed and tried again with BSA (4% BSA blocking for 1h RT and 1% BSA incubation with primary Ab 4°C ON and 2h RT with the secondary Ab) and worked very fine! How is possibile that BSA is better than a Normal Goat Serum? Did I make a mistake mixing it with FBS?
Thank you!
Serum contains number of antigenic molecules that may interact with Primary Ab as well as Secondary Ab and generates nonspecific background. Another possibility is that the you used goat serum also for blocking purpose and your secondary antibody is raised in goat. Hence, the similar source (goat serum and Sec Ab in goat) might have generated interactions between goat serum Ag with Secondary Ab. This could have caused lots of background as you stated.
On the other hand, when you used BSA (Bovine serum albumin), the antigenic molecules source from serum (bovine as well as from goat) have been eliminated and nonspecific interactions was eliminated. Thus, you got expected results.
Best
You should only use a single serum in your blocking solutions, generally, this should be from the host animal of your secondary antibody (in your case goat). There isn't a single serum concentration that will work best in all cases, but I've never used a concentration above 5%. I also wouldn't recommend mixing serum from two different species.
That being said, BSA can also be used in the blocking solution. If that worked for your antibody, I would just keep on doing that.
Daniele it is often recommended to block with a serum of the species in which the secondary Ab is made. Occasionally you can prefer BSA even if the quality of saturation could be less than the former.
Marika Crescenzi thank you! as I mentioned, in the first attempt I used a blocking solution containing 10% FBS and 5% Normal Goat Serum (since my secondary antibodies are raised in goat) but it was awful. Then I tried with BSA only and worked pretty well.
John Hardy Lockhart thank you! So mixing FBS and normal goat serum was not a good idea. I will try with 5% normal goat serum only.
Shail K Chaube thank you very much for you answer!!
Dear John Hardy Lockhart , I tried again to do an IF of different proteins in cells transfected with the respecive siRNA in order to see whether the signal I am seeing is specific or not. I incubated cells for 1h at RT in blocking solution containing 4% normal goat serum (NGS) and 0.1% saponin, then overnight with primary Ab at 4°C in 1% NGS and 0.1% saponin and 2h RT with secondary Ab raised in goat in 1% NGS and 0.1% saponin. Results, the signal is not specific at all for all Ab tested and looks exactly the same for all Abs. I do not understand why all these problems when incubating cells in normal goat serum. I am goint to use BSA as previously did and forget NGS.
That is unfortunate. If the BSA blocking works, I would do that instead. I'd also recommend contacting the manufacturer of your secondary antibody to let them know about the issues you've had.
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