Dear All,
For new/nerver-used antibody and no recommend dillution buffer to use with in datasheet, Should I start trying to dillute it with TBST alone first?
I know that dillue antibody in blocking buffer (either notfatmilk or BSA) is helping to reduce background. However, I heard someone complained that add milk or bsa in primary antibody dillution can reduce protein signal, espicially for low sensitvity protein. Not sure if It's true.
Thank you
Cheerio,
Dear Asa Wu,
Diluting your antibodies in a 5%BSA solution is the option to go for it.
You might store your diluted antibodies in -20 if you want to reused many times.
Best,
Hamadi
I agree with Hamadi for using BSA to dilute the antibody. For storage, I would recommend 4 degree with sodium azide as preservative. In my experience, antibodies stored in BSA + azide can be reused for about 1 month easily.
For most antibodies it's also okay to use non-fat milk, which is cheaper. As a rule of thumb, if the epitope of interested is in your dilution buffer, it will absorb your antibody, which is of special interest for antibodies against phosphorylation or example.
If you want to work with the antibody more often and your results are not as expected you should check out the optimal buffer conditions (some antibodies prefer BSA instead of milk or otherway round, some others don't like Tween or only work in PBS and not in TBS and so on). Blocking substances in the dilution buffer is a good idea generally and for most antibodies standard dilutions in blocking buffer are just fine.
For storage it's also a good idea to add some protein (BSA or milk) to stabilize the antibody and NaN3 as said above.
And don't forget to block your western blot membrane before adding primary antibody! Adding blocking solution with your primary antibody is not enough.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques