We want to characterise some brain regions in an insect using an anti-synapsin protein. Most protocols use Normal Goat Serum to dilute the antibodies and as a blocking solution, but it seems to be difficult to get here in Mexico. There are alternatives, like BlockAid, a proprietary formula that is better at blocking unspecific binding (according to the manufacturer). The problem is that I haven't found a single paper where BlockAid is used in insects with our antibodies. Has anyone used these solutions? Are they really better? What are the possible drawbacks?
We have good results using antibody diluent from Zytomed Systems on human and mouse, although we did not yet use it with insects. This should be available in Mexico if you contact the company.
Hi Ricardo,
I assume this is for immunofluoresence? 1% BSA in PBS is very common and citable for blocking. I noticed on Thermofisher today that you can buy a 10%/10X solution of BSA/PBS. I typically buy BSA from Sigma and make the 1% solution in PBS, but the premix might be cheaper.
https://www.thermofisher.com/order/catalog/product/37525
I bought NGS recently to be a good boy and use the serum that my secondary was raised in. I didn't find any difference compared to BSA. That's just my experience with my tissue though (pig).
best of luck!
Rich
Have you tried cold water fish skin gelatine as a blocking Agent? Its an old technique, not so well-known nowadays. This should also be available in Mexico.
Thanks for your answers.
Yes, it is for immunofluorescence. BSA is available here, so we'll give it a try. I didn't know about the fish skin gelatine. I read some protocols and it seems quite simple, and cheaper than NGS
There are a number of considerations to take into account when using your blocking reagent. Using whole serum looks attractive on paper as it contains several proteins, yet the major protein is serum albumen. If you use whole serum, there will also be IgG's in there, and it is for this reason that people use whole serum from the same species that was used to raise the secondary antisera [think what would happen if you were to use the whole serum from the same species in which your primary antisera were raised]. In the past I have used secondary pig antibodies, and normal pig serum was easily obtained from an abattoir. Most of my secondary antisera are made in goats and I use normal goat serum. For many antisera BSA will work just as well, however I never try that as I have many neuropeptide antisera in which BSA is used as the carrier protein and hence my primary antisera may contains anti-BSA IgGs. If anti-BSA IgG's were to become a problem other blocking reagents like the one suggested here (fish skin gelatin) or non-fat milk powder may work as well.
Thank you for your answer, Jan. I forgot to mention that our secondary antibodies are goat anti-mouse.
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