I always had very good experience with Cell SIgnaling antibodies if you exactly follow their recommendations for the respective antibody.

As others have mentioned,blocking conditions can make some difference, but if  CST recommends BSA stay with BSA. Milk blocks better, but for some phospho-antibodies it could only make things worse.

Refer to CST protocol for your antibody. Block for at least 1h and primary overnight at 4degrees. If u are using a strong ECL solution, washing will be critical. I have had a cloud like signal throughout the blot, if not washed properly. 3x 10 minutes or so should be good.

Good Luck.

Are you diluting your block in PBS with a mild concentration of a detergent such as Tween20?  I use milk, BSA, or sometimes gelatin, dissolved in 1 x PBS, with 0.05% Tween20.  As suggested above, make sure you wash at least 3x's  after incubation (I do 3x's 10-15 min., on a rotating plate, with PBS/Tween).  For 2nd, from my experience, I NEVER use any 2nd diluted under 1:10,000.  I sometimes go as high as 1:20K or 1:25K (for an hour rt).

Oh.. also you need to be careful about "drying" your membrane.  You don't ever want to lay it down on a paper towel, as you will pick up lint from the paper towel that will contribute to your background.  Pick the membrane up carefully with tweezers at the very edge, and only dab the very bottom or bottom corner on the paper towel to remove excess moisture.  You don't want the membrane 100% dry anyway.  

I've had really good luck with Cell Signaling phospho-specific antibodies.  I use 1% BSA in TBST (I'd stay away from milk with a phospho-specific antibody b/c of the possibility of phosphorylated proteins present in the milk, especially if Cell Signaling advises using BSA).  I use TBS instead of PBS for phospho-specific antibodies (I may be paranoid, but I've always wondered if the phosphates in PBS could give nonspecific interactions).  I'd also lower the concentration of your secondary antibody (I usually use a 1:10,000 dilution of a 1 mg/mL stock).  There's a slight possibility you can salvage the blots you have by just washing the heck out of them--I've let blots wash for several nights at 4 deg C & still had good signal--it could help reduce the background enough to at least see the bands, if not give you a pretty blot this time around.

Hi, Kira, you laready have a couple of excellent advices. I will add my 2 cents. In some case BSA in teh blocking buffer may block anibody binding to the epitope. This event is dependent form anibody types.

 Actaually, to avoid blocking step and to increasing prtein binding to membrane you can completely dry membrane  after tranfer,. This step alos may reduce backgorund.

However, your shouldmnever dry mebrane during incubation of the antody or during washing. In thsi case you will have a very high background.

Good luck!

Anyone with experience can give you excellent suggestions about what the problem(s) might be, but because you haven't described any of your controls our  suggestions are much like a fishing expedition. This is why controls are always necessary  as reagents expire, new antibodies often require optimization, power box failures, poor transfers, type of membrane (PVDF?), etc.

Dear Kira,

Already you probably have a better idea for how to avoid non-specific signaling while targeting phospho proteins  when using BSA in the blocking buffer. I handle a large number of phospho proteins in WB almost everyday and I prefer Li-Cor Odyssey blocking buffer, Li-Cor Biosciences, or EIA Aquablock buffer (PP82) from East Coast Bio. These are fish protein based blocker and containing PBS.

Though I must admit that the endpoint detectin that Ii use is not the ECL, it's Li-Cor Odyssey IR scanner that recognizes either 680 or 800 nm conjugated secondary antibodies. The beauty of those fish protein based blocking buffers are that there is least possibilities for cross reactivity with the primary antibody and for photspho signal it reduces non-specific background signal significantly.

You can certainly think of using those blocking buffers to solve your problems.

Good luck!!

If you double-check the recommended protocol it probably indicates that you should block for 1hr in 5% milk and incubate the primary in 5% BSA. 

I agree with Mark; block with 5% milk in TBST (I use 20 minutes) then use TBST 3% BSA to dilute primary. Not all BSA is suitable for western; I use Sigma cat #7906, and it works quite well. I also agree with Monica, increase your washes or washing time (I usually do I hour)

Hello Kira, 

Firstly I'm using Licor instrument to detect my protein of interest which does it based on the wavelength of my specific secondary antibody used against my primary,don't know if that will make any difference to your study ( can imagine not all labs have this facility and same time ECL is more reliable and less expensive).

After the wet transfer, I blocked my nitrocellulose membrane in 5%BSA (Sigma) in TBST for 1 hour and later used 1:2000 of primary antibody (optimised concentration) diluted in 5%BSA in TBST.  After overnight incubation of the primary antibody, membrane was washed 3x10 min  and probed with fluroscent secondary antibody (1:15000 or 1:20000) diluted in TBST and incubated in dark at room temperature for 2 hours on a shaker  by covering the membrane in a foil ( I presume you use a petridish container to store your membrane). And finally I wash off the excess unbound secondary with wash buffer for 3x10 minutes and ready to analyse on Odyssey software( but I have never used ECL detection method).

Key thing to remember at the start of your experiment is to use higher amounts of protein sample ( I've used upto 40ug of tissue sample) to detect phosphorylated protein as phospho protein may not be in excess in most cases,so you will need more protein starting material to see any bands in the end.  This. Protocol has worked for me so far  and although I don't see a nice thick band instead I see a faint band for phosphorylated protein in my samples and have compared that against b-actin to make I have added the right amount of samples.

hope this helps

good luck with your experiments 

Seshadri 

BSA is an inferior blocking agent to blotto (5% milk). The problem when performing phospho antibody Western analysis is that milk contains numerous phospho proteins, including a very large concentration of Casein. As a consequence, using milk during the primary antibody steps  often times eliminate the signal.

What you should do is:

1: perform the initial blocking step using BSA. 

2: dilute your primary in BSA

3: After primary incubation, wash membrane 3x vigorously using high volume washes

4: Now block a second time using 5% milk.

5: dilute your 2nd Ab using 5% milk.

6: Wash membrane 3x vigorously using TBST

This allow your primary to bind without interference from milk, but still provides  proper blocking in the secondary steps- when its most important.

Using quality secondaries is also helpful. Not all companies make clean secondary. ( Southern biotechnology a +++ secondary company)