Hello, Everyone!
I currently start doing western blot experiment, but I am not really sure how much should I add primary antibody into 20mL of 1% Skim milk.
Here is what I am doing:
* I make 1% Skim milk by adding 0.2g of Skim milk powder into 20mL of TBS-T.
* I want to add primary antibody (LC3B) which was purchased from Santa Cruz. It was mentioned in the guideline that each vial contains 200µg IgG2b kappa light chain in 1.0ml of PBS with
It will be better, if you share me catalog number. Generaly, antibody dilution used according manufacture recommendation.
When they say a 1:100 or 1:1000 dilution, for a liquid into a liquid is volume by volume.
So... 1:100 is 1uL into 100uL and a 1:1000 is 1uL into 1000uL
So... for 20mL a 1:100 dilution is 200uL of antibody a 1:1000 dilution is 20uL of antibody.
you could start off with a higher dilution say 1 in 1000. That is 1mcL(micro litre) of antibody in 1ml (1000 mcL) of 1% skim milk. So for 20ml of blocking buffer, use 20mcL antibody. If it doesn't work, go for a lower dilution.
Does it answer your question?
Shen-An Hwang Nikhitha John Thanks so much for your explanation. However, does it means that we don't need to care about the stock concentration mentioned on the antibody vial, right? (Here: 200µg IgG2b kappa light chain in 1.0ml of PBS).
Sokviseth Moeng Usually the stock concentration on the vial doesn't matter if the data sheet has provided an optimal dilution range to use. In your case 1:100 to 1:1000. You need to standardize it based on your experiments.
Nikhitha John Thanks so much. I understand now. So the stock concentration mentioned on the vial is not the matter. We just dilute the amount of antibody from the stock with the diluent as recommended in the data sheet.
For example, if it is 1:1000 --> I need to add 1uL of antibody stock to 1,000uL of diluent. Am I getting it right?
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