It is better using 2% BSA in PBST or 5% milk in TBST. If with 5% milk you don't see the band, use 0.5% milk for the primary. TBST alone can give you too much background. Anyway the choice is based on your primary: house keeping gene I would go with 5% milk, low expressed protein with 2% BSA or 0.5% milk.

Hi, From my experiences I would like to say each antibody has its own conditions which are depend on the maker ( a company). I recommend to go with the company protocol that is provided with your antibody.

Good luck,

For western blotting you better incubate the membrane for blocking in 5% non fat milk or skim milk (prepared in PBS buffer) {you can use 3% of BSA as well}at 4 degree with gentle shaking for overnight. Then next day wash 2-3 times with PBST then incubate in primary antibody for atleast 2 hours..then again vigorously washing with PBST 2-3 times then incubate in secondary antibody..

Dear Khairul,

Sabrina's answer seems very comprehensive and well researched so I believe you had already got your answer. Primary and secondary antibody dilutions in blocking buffer is the preferred condition for competitive blocking for a less noisy and clean background.

Depending on your endpoint WB detection system the choice of blocking buffers may also vary. For very sensitive IR based detection system (Li-Cor Odyssey), proprietary formulation of blocking buffers are available from the respective company or you can use a fish protein based EIA blocking buffer in PBS, pH 7.4 (eg Aquablock, PP82 from East Coast Bio). Since primary antibodies are usually developed in mammalian system fish protein based blocking buffer actually creates very less non specific signal and enhances the quality and precision of the detection.

thanks everyone for the answers and recommendation.

today i managed to get my very 1st time image of protein expression on my film with clean background .(see attachment) but the problem is, why I can't see the protein marker? isn't normal or anything that i can do to troubleshoot it. thanks.

Hello Everyone. In my experience always disolve ABs in TBST 5% skimmed milk. Whole milk develop high background on the membrane.

Did you use a biotinylated protein ladder or prestained?

Dear Kellie Bowen,

I used prestained protein ladder (Spectra Multicolor Broad Range Protein Ladder, Thermo Scientific). The protein ladder only visible after the membrane being exposed more than 10 minutes but my protein expression became too dark (very high intensity).

You can use this if your doing Chemi.

http://www.cellsignal.com/products/experimental-controls/7727

I aliquot the vial out (6ul vials) when I get it and use 5ul. It comes with the biotinylated secondary. Use it at 1:15K along with your other secondaries or it won't work.

If you can see protein marker on the membrane then It is normal that you could not see protein marker on the film for short exposure time.

All excellent answers. I agree.... keep the milk in all stages until the final washes. If you have a really difficult antibody then increase the milk up to 10% or use a different blocker and play with the concentrations of Tween and maybe salt as well. Its very rare I've had to do that and its usually easier to find an alternative antibody. Here's the protocol I've finally settled on after 34 years of doing Westerns. Transfers are done onto Immobilon PVDF membranes

Block 1hr PBS pH 7.4, 0.2%Tween, 8% skimmed milk (PBSTM)

Primary 12-18hrs PBSTM

Wash 3x 5mins PBSTM

Secondary (peroxidase) 2hrs PBSTM

Wash 10x5mins PBST (probably excessive.....5 can be enough but I do usually get nice clean blots. I sometimes leave the blots washing in the final wash for a couple of hours

Develop using ECL Prime and Hyperfilm

Overnight in primary is definitely much better than the 2 hours in the original protocols but only if you have a clean antibody (I'm lucky enough to have an Amersham Processor Plus so my method is designed to run overnight so the blots are ready to go on film first thing in the morning and the results analysed before immediately setting up the next gels).

Regarding MW markers..... I only used a prestained marker to tell me if the gel has run OK and transferred. I mix this (to save using an extra lane) with Invitrogens MagicMark which shows up on the film no matter what species of antibody has been used. Slightly expensive but I only need run a few ul of a 1:10 dilution for it to show up. Unlike prestained markers it also gives you an accurate molecular weight.

None of the above is foolproof and I still have occasional disasters but thats westerns for you!

I will recomends the TBST. I got interesting band if i use TBST for primary and secondary antibody incubation. Good luck