I was doing a phosphorylation study of Tau using tau-1 antibody from Millipore, I am having trouble getting bands... on contacting the company they asked me to change the blocking solution to the 1% serum of secondary antibody source in PBS and include Triton-X 100. I had been using the BSA solution in TBST which has tween-20. Basically triton-x 100 and tween both being surfactants should have similar properties rite, or is any one of them superior above the other in western blots. Please do pass me ur ideas.........
Also in the monograph they stated the final conc as 5ng/ml (1:200,000) for d monoclonal antibody, and they again told me to use 1:200 conc saying it might be 5ug/ml of final concentration. Has anyone got experience on Tau-1 or any related ideas u coud pass on to me, please pass it on.
Thanks
Sanjaya
phosphorylation(ATP-ADP) and PBS( contain K2PO4, KHPO4) are difference.
BSA and MILK.
Hi!
Have you tried to prepare your samples without mercaptoethanol? I have experienced that some antibodies recognized better the proteins when they have not been reduced. For p-ser and p-thr it is better to use BSA. Milk causes a LOT of crossreaction with the membrane. I will do what the company says, just to prove them wrong (= and you can get a replace antibody/coupon... good luck
Luisa
P.S. Could you stain your gel with commasie after the transfer to the membrane? That will tell you if the transfer was complete.
@Jia Ping- Seems u did not get my point, anyways thank you.
@ Luisa- So far I have not tried to prepare samples without reduction--I have been using DTT in all samples. May be I should give it a try if other methods do not work out since I recently collected many samples already.
I have been thinking about a commasie staining to see if d transfer is complete.
Thanks again
I do experiments, very close to yours. Try to block with 5% milk in TBST, wash 3x for 10min, than use prim.AB (deluted in TBST, 4°C over night). You have to be shure ti have no milk (especially the phosphorylated casein in milk) is in you AB- solution.
Are you talking about total Tau1 antibody-''Anti-Tau-1, clone PC1C6'' from Millipore. In that case I think the company is mixing up the applications. The protocol which u mentioned (Triton and serum blocking) is commonly used for immunostaining and it is stated in their online datasheet (5µg/mL antibody) and for westren they suggest 5ng/mL antibody.I dont think Triton is too good for membrane based techniques, May be 0.03% what they suggest works.
Currently what is ur problem? lot of background / nothing at all??? If your problem is with total Tau antibody then it cannot be a background problem due to milk (I feel for total proteins MIlk works better than BSA). Can U describe ur current procedure. What cells are u working with?
About coomassie staining of gel, I could never see a complete transfer unless I am loading 5-10µg / lane, eventhough western works fine for very low expressing kinases. The best thing would be to stain ur membrane with Ponceau to see that there is sufficient protein on the membrane.
Good Luck
Manoj
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