A) Why would one prefer 1XTBST over 1XPBST or vice versa?
B) If one is detecting phospho proteins, does it matter which one you are using?
I have been doing Western Blots for a very long time but have never come across a proper explanation for the use of 1XTBST over 1XPBST or vice versa.
for phospho proteins you have to use TBST
personaly I'm used to do all my WB with TBST and all my antibodies are working like that !
Ozgür is right. It depends on your antibody. But much better is to wash tree times. Put as much volume on the blots and remove at the end of each step as much as possible. Washing is nothing else than diluting your reactants. Time is not that important but the number of washing steps.
If you have problems with potential cross reactions or background, increase the NaCl concentration for washing (and even sometimes for incubation) up to 0.3 ... 0.5 mol/L.
I agree with Laurent: I usually use TBST for phospho proteins and PBS for others but as Ozgür said, it mostly depend on your antibody. Also, datasheets often inform about using BSA or milk and it might be very important sometimes...
I always used TBST and 5% skimmed milk for my PhosphoWB. We have made 5 different Phospho-specific antibodies and it always worked out very well. We even used something like 1-2% milk for washing if we had trouble with the background. It depends a little bit on what sensitivity you need and how much sample you have.
I disagree that you have to use TBST for phosphospecific antibodies, because I used PBST for ELISA and had not sensitivity problems. It would be a very unspecific antibody if it can be blocked it using a phosphate buffer. I always suggest that you have to try what works best for you. To my opinion were is no rational assay design if you use antibodies, because we experienced a lot of funny things e.g. that antibody specificity changes when using different blocking solution like BSA vs. Casein.
general rule of thumb. TBST with total antibodies and PBST with phospho-specific antibodies
I found no differences in TBST and PBST washing buffer. The blocking solutions matter, 5-10% skimmed milk, Li-Cor blocking buffer, BSA, chicken serum etc.
I never thought TBST or PBST make a big difference as washing buffers, yet I agree now with previous posts that it depends on your antibody. I found that using PBST in one blot I did recently gave lower background noise than TBST. Also you might want to try different Tween concentrations as they increase your detection level in some cases.
TBST better especially if you are using an Alk-phos conjugated secondary antibody. Like the others that have commented before me I also use 5% skim milk and sometimes 1-2% Tween if background a problem.
Washing is advised after both the steps. TBST is better, because of the presence of Tween it removes all other proteins except specific antibodies.
It depends of the antibody ... that's it! Follow the indicated procedure if it is a commercial antibody. In some very rare cases the interaction between antibody and the targeted protein needs additional component such as Ca2+ ou Mg2+.
If you use alkaline phosphatase coupled to your detection antibody, phosphate will inhibit the color reaction! However, it appears that some antigens do not like washing with TBS so much and thus washing with PBST might be more gentle for the coated protein. In this case you should use peroxidase coupled detection reagents. Nevertheless, I never really experienced real problems with TBST as washing solution.
Use TBST for washing PVDF blots and it works just fine.
I guess that the original reason for using TBS instead of PBS is that phosphate interferes with alkaline phosphatase based detection.
With HRP or fluorescent secondary ab conjugates that's not a problem.
Same as above TBS-T (1.4% as it is a tiny bit more stringent). Then depending of your protein of interest 5% milk in TBS-T or BSA for the blocking step. I personally never used PBS in WB.
Good luck.
- Badges
- Science method