PBS vs TBS for western blots - Background problems (May/07/2007 )
Hello everyone
Does anyone know if TBS-Tween is better/worse/no different than PBS-Tween for western blot washing?
I have nice, clean western blots for detecting my protein of interest in transfected cell lysates but now need to detect it in tissue lysates. This has meant an increase in primary antibody and the inevitable background issues.
At the moment I am using PBS with 0.1% Tween 20.
Is switching to TBS going to solve my problems or is there a more effective method?
I only have a limited amount of tissue lysate and can't afford to waste it all on optimisation.
Hi There,
Depending on your protein, sometimes phosphates can complicate. In this case, Tris works better. But, I have noticed after running about 3000 western blots and others sometimes the difference in your problems can be attributed to the blocking agent. Often, the secondary IGG may react with BSA and the primary with milkprotein. So, I have avoided the problem all together by always running my block in BSA-TBS-T, primary in same, and secondary (after 3x20 min washes) with milkblot (5% in TBS-T). They turn out well everytime. Secret is to wash, wash, wash!!
Good Luck!
Does anyone know if TBS-Tween is better/worse/no different than PBS-Tween for western blot washing?
I have nice, clean western blots for detecting my protein of interest in transfected cell lysates but now need to detect it in tissue lysates. This has meant an increase in primary antibody and the inevitable background issues.
At the moment I am using PBS with 0.1% Tween 20.
Is switching to TBS going to solve my problems or is there a more effective method?
I only have a limited amount of tissue lysate and can't afford to waste it all on optimisation.
[quote name='biokmst' date='May 11 2007, 10:21 PM' post='97446']
Hi There,
Depending on your protein, sometimes phosphates can complicate. In this case, Tris works better. But, I have noticed after running about 3000 western blots and others sometimes the difference in your problems can be attributed to the blocking agent. Often, the secondary IGG may react with BSA and the primary with milkprotein. So, I have avoided the problem all together by always running my block in BSA-TBS-T, primary in same, and secondary (after 3x20 min washes) with milkblot (5% in TBS-T). They turn out well everytime. Secret is to wash, wash, wash!!
Good Luck!
Hi biokmst.
What you said about the primary Ab reacting with milk proteins was very interesting.
Can you tell me a little bit more. I mean if I do the blocking with BSA-TBS-T, the primary with BSA-TBS-T and the secondary with 5%milk in TBS-T is OK?
I checked in my lab and for the time we don't have BSA but a blocker solution of Biorab (TBS-T casein, i think) will it be OK with that instead of BSA-TBS-T?
And just another question...With what kind or brand primary antibodies did you noticed that reaction with milkproteins?
Thanks a lot in advance, I hope you can find the time to answer me.
It will be great help because I'm getting a bit out of ideas with what is going wrong with the protein I'm looking.
Thanks again 
Does anyone know if TBS-Tween is better/worse/no different than PBS-Tween for western blot washing?
I have nice, clean western blots for detecting my protein of interest in transfected cell lysates but now need to detect it in tissue lysates. This has meant an increase in primary antibody and the inevitable background issues.
At the moment I am using PBS with 0.1% Tween 20.
Is switching to TBS going to solve my problems or is there a more effective method?
I only have a limited amount of tissue lysate and can't afford to waste it all on optimisation.
i think whether TBST or PBST is dependent on which kind of membrane is applied. NC membrane is washed by PBST, and PVDF membrane is by TBST
Hi,
I 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.
Jaakko
I would suggest just use PBS (ph 7.0) for all washing steps in the WB protocol. i have run 100s of blots & to my experience washing in excess PBS(~70ml) for 10mins three times...excludes all background signals.
also try using your pri AB raised in rabbit or mouse. if your using goat u will have dark days ahead 
hope it helps
Raj