usually, for western blot, mAbs are diluited in TBS-T at the final concentration of 1 ug/ml...

it depends on your antibody...check the mAb data sheet for more informations...

usually, for western blot, mAbs are diluited in TBS-T at the final concentration of 1 ug/ml...

it depends on your antibody...check the mAb data sheet for more informations...

thanks a lot Nicola, actually, I didnt apply western blot experiment yet, but I want to ask what did they mean by: the dilution is 1:200 while the primary antibody stock is 200 μg, and what is the appropriate final volume of the primary antibody solution that is enough to soak the nitrocellulose membrane, is it 5 ml or 10 ml? cause I read in an western blot procedure:

" Incubate with primary antibody at appropriate dilution in TBST + 0.5% milk

for at least 1 hr at RT. Use 3-5 ml " can you explain this to me?

if the primary antibody is 200ug/ml then to make a 1ug/ml stock (or a 1:200 dilution), dilute it in TBST+0.5% milk i.e. to make 5ml of the dilution, add 25ul of the primary antibody to 5ml of the blocking solution (TBST+milk)

We also use a 1ug/ml final conc of antibody but, as Nicola said, check the data sheet for their recommendations on the dilution to use. It may take some optimisation though..

Sometimes, when the mAb is poorly concentrated, we use a trick: a small volume of Ab and blocking solution on the membrane wrapped in a plastic bag (using a hot sealer). In this way we can use less than 1 ml to cover the membrane and incubate it...

In addition, it is a good antibody, you can diluite it more (i.e. 1:1000, so, more or less 0.2 ug/ml)...but, again, it depends on your data sheet...

And also...if you use 5 ml of mAb solution, you can re-use it 3-5 times (remeber to add 0.01% NaN3 to prevent bacteria contamination and store it at 4°C)...

Hope it helps...

If you don't have a hot sealer you can cover your membrane with just enough antibody solution and then put a piece of parafilm on it (it has to cover the whole surface). You can then incubate for as long as you like, even overnight at 4degC.