Blocking before hybridizations

[Monique]

I was wondering how the blocking of a sample works with serum before hybridizing it with an antibody.

I mean, you want to block everything aspecific so that the antibody doesn't bind everthing, but with that it blocks things that the antibody is supposed to bind right?

Or is it all about affinity, the antibody should have enough affinity to bind no matter that it is blocked..

And exactly what is the blocking agent in serum? Albumin? And why does it block?



I'm braindead after waking up at 6 and coming home at 7, eating and after that studying and then going to sleep around midnight..

 

[iansmith]

The blocking solution is made with a blocking agents which is could either be serum albumin, skim milk or tween. The blocking agent will cover the protein or surface with a slight coat and block unspecific binding but it will not influence the specific binding.

I think the pH, salt concentration and concentration of blocking agent will influence the stringency of the binding. pH and salt concentration probably need to be stable because buffers are always used.

 

[ravenprp]

Blocking before hybridizations is an important step in many molecular biology techniques, including immunofluorescence and Western blotting. It involves treating the sample with a blocking agent to prevent non-specific binding of the antibody to the sample during the hybridization step.

The purpose of blocking is to reduce background noise and increase the specificity of the antibody binding. Without blocking, the antibody may bind to non-specific proteins or molecules in the sample, leading to false positive results. By blocking these non-specific binding sites, the antibody can more accurately target and bind to its intended target.

The blocking agent used in serum is typically a protein, such as albumin or casein. These proteins have a high affinity for binding to surfaces, such as the sample or the container, and can therefore block non-specific binding sites. Additionally, they can also bind to any free-floating antibodies in the sample, preventing them from binding to non-specific sites.

It is important to note that blocking does not affect the binding of the antibody to its intended target. The antibody should still have sufficient affinity to bind to its target even after blocking. However, it is important to optimize the blocking conditions to ensure that the antibody can still bind effectively.

In summary, blocking before hybridizations is a crucial step in molecular biology techniques to reduce background noise and increase the specificity of antibody binding. The blocking agent used, such as albumin, works by binding to non-specific sites and free-floating antibodies, without affecting the binding of the intended antibody.