I am working with chemiluminescence substrate in western blot. Some of researchers suggest PBS (phospate buffered-saline)/PBS-Tween 20 for washing the membran after blocking and primary/secondary antibody incubation instead of TBS (phospate buffered-saline)/ TBS-Tween 20. Which one is most suitable?
Both are OK, but we use TTBS in a regular basis . TPBS is cheaper though.
TTBS > TPBS for blots especially if there are phospho-proteins to detect.
I'm not sure if this will help, I just remembered that the choice between PBS and TBS depends on the protein's of pI (isoelectric point): 1XPBS buffer is ~pH 7.4, where most proteins have pI at this pH level so they conform to their no net or neutral charge. Antibodies have pI near approximately 8 to 9, thus using TBS with pH range of 7-9.2 will conform the antibodies at no net neutral charge. With this information, if TBS is available, it might be better to be used because it can buffer wider range of pH, making all the proteins present in your blots maintain their no net charge conformation.
Additional information, borate buffered saline has a pH range of 8 to 10, so proteins with isoelectric points within this range can be processed using this buffer. I hope this information will help.
Sincerely,
JP
Another point for your consideration is the conjugated enzyme to your antibody, since PBS/TPBS could interfere with alkaline phosphatase. TBS and PBS both work ok for HRP.
Dear James, John and Carlos,
Thank u all for your help. It will be helpful for my studies.
I use TBST for most of the washing, but the very final washes before the detection I use TBS. This is probably superstition, but ancient lab tradition tells me that in some cases the tween can interact with the chemiluminescent reagents and thus produce worse quality signals.
- Badges
- Science method