Washes for Western, PBS or TBST? - (Mar/13/2006 )

Hi I'm just curious if people do their post primary antibody pre-detection washes of a blotted PVDF membrane in PBS or TBST, and if one rather than the other, why?

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hey cwong

if you're looking at phosphorylation state, you HAVE to use TBS (the phosphates in PBS will screw up your results)

this is the main difference of which I am aware

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hi,
aimkins in our lab we use, phospho ERK, jnk n p38 antibodies, every body uses PBS.
I wondering about ur answer, if possible please make me clear in this concept.
thanks

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those are the same Westerns that we do. I was told that TBS would screw it up and give you high background, as well as potentially interfere with your results.

I do not have a reference; however I have heard it from several people. Perhaps it's an old-wives' tale?

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I use 6x TBST before adding my secondary, and 5xTBST then 1x PBS before detection. I was told that the tween can give high backgrounds and to wash it off with PBS before beginning the detection.

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We wash 3X's in TBS-T and 1X PBS and it works fine!

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Hi:

I use PBS. I will look for phosphorylation state soon, so i'm interested in what you said. I also would like to ask if somebody has used anti phosphoserine antibodies to detect phosphorilated proteins. If so any recommendations? Is it also possible to quantify and compare phosphorylation state using this antibodies
Thanks

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You have to remember that PBS is a phosphate-buffered saline and TBS a tris-base saline. Phospate groups in PBS can interfere with anti-phosphate antibody binding to your potentially phosphorylated protein and will bind all of the phosphate groups in the saline instead of your protein phosphorylation sites.
Also, milk is not used for blocking when anti-phospho antibodies are used due to phosphoproteins in the milk which will compete for the phosphorylation with your potentially phosphorylated proteins.

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and don't forget that pbs will inhibit alkaline phosphatase. so you want to avoid pbs if you are using ap conjugate for detection.

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the main difference is that TTBS contains the blocking agent Tween which increases specificity during Ab incubation whereas PBS has no blocker;

if you really like to decide which buffer is to prefer you have to test both buffers for each Ab;

one theory in perferring PBS for phospho-state analysis is that the high concentration of PO4/3-lowers the pressure of the hydrolization of phosphoryls from polypeptides;

nevertheless, we have decided to use routinely TTBS although we analyze a lot of phospho-proteins

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