Primary Antibody: Is it necessary to dilute antibody in 5% skim milk?

[new2science]

Hi There,

I am confused about what I should dilute my primary antibody in. I have been using 5% skim milk in TBST when I dilute my primary antibody and secondary. But I know someone in a different lab uses just TBST to dilute their primary antibody with NO skim milk at all and it seems they are able to visualize their bands better that using the skim milk method.

I do block my membrane in 5% skim milk for one hour before incubating my primary antibody overnight, so I'm thinking that it isn't necessary to dilute my primary antibody in 5% skim milk.

Does anyone have suggestions?

[manders77]

Hi New2Science,

It's not necessary to dilute your primary antibody in blocking solution. I used to do that, but now I just use PBS-T (or even PBS), and I don't have any problems with high background. It really just depends on your antibody. As long as you have a good primary antibody and you have blocked your membrane well, you should be fine with omitting the blocking reagent from your primary antibody incubation.

-Manders

[Warthaug]

As manders said, it depends on your antibody. You can often exclude the block in the antibody solution, but if you have background issues, maintaining block in your antibody solutions often helps.

Many of us keep it in, more out of lazyness than anything.

Bryan

[new2science]

Hi Manders and Bryan,

Thanks for your advice. I will try my experiment without the blocking solution and I will see if this works.

Thanks again!

[new2science]

Hey guys,

I just wanted to say thanks for your opinion! I tried diluting my antibody with skim milk and without skim milk. Both methods worked!

Thanks again,