there are a few common things to consider to optimize looking at phospho proteins.

1. most important thing is to process your tissues with phosphotase inhibitor cocktail.  I usually use Halt Protease and Phosphotase inhibitor from Pierce at 1:100 concentration but many companies have this product.  

2. When you are preparing your sds samples for loading, make sure that the samples remain on ice before you add your sample buffer.  Keeping the samples cool helps prevent phosphotase enzymatic activity that can occur at room temperature.  Once, the sample buffer had been added you are ok.

3. When you are performing your blotting procedure make sure to incubate with your antibody in 3-5% BSA and not milk.  Milk contains caseins that have phosphotases and you want to avoid this.  Block your blot in milk, add your primary in 3-5% bsa, and then add your secondary antibody in 5% milk.  

4. If you are using performing standard chemilumenence, use HRP conjugated secondary antibodies and not alkaline phosphotase secondary antibodies.

5. Phospho proteins are generally more sensitive so avoid freeze thaw cycles of your samples.  This can cause degradation of your signal.  

6. If your signal is low, you can always load a little more protein in your gel.

7. If you are looking at both phospho and total protein on the same blot, look at the phospho form first before stripping.  Phospho proteins do not respond well after a stripping procedure.

good luck!

there are a few common things to consider to optimize looking at phospho proteins.

1. most important thing is to process your tissues with phosphotase inhibitor cocktail.  I usually use Halt Protease and Phosphotase inhibitor from Pierce at 1:100 concentration but many companies have this product.  

2. When you are preparing your sds samples for loading, make sure that the samples remain on ice before you add your sample buffer.  Keeping the samples cool helps prevent phosphotase enzymatic activity that can occur at room temperature.  Once, the sample buffer had been added you are ok.

3. When you are performing your blotting procedure make sure to incubate with your antibody in 3-5% BSA and not milk.  Milk contains caseins that have phosphotases and you want to avoid this.  Block your blot in milk, add your primary in 3-5% bsa, and then add your secondary antibody in 5% milk.  

4. If you are using performing standard chemilumenence, use HRP conjugated secondary antibodies and not alkaline phosphotase secondary antibodies.

5. Phospho proteins are generally more sensitive so avoid freeze thaw cycles of your samples.  This can cause degradation of your signal.  

6. If your signal is low, you can always load a little more protein in your gel.

7. If you are looking at both phospho and total protein on the same blot, look at the phospho form first before stripping.  Phospho proteins do not respond well after a stripping procedure.

good luck!

It might also help to use an enhanced sensitivity substrate, this is what we usually do (SuperSignal West Femto Maximum Sensitivity Substrate from Thermo Scientific). It works very well and gives strong bands by chemiluminescence.

Good luck!

Not a lot to say after the answer of Brandon :) but to be 100% sure I prefer to use BSA for the whole experiment (including the blocking step and the secondary antibody step). For the low signal, there is also the solution of using some enhanced HRP substrate that will be able to detect pg of proteins. Generally, the name is something like "ECL Plus". Good luck !

Hi Brandon,

Thanks for the details reply

Actually I followed almost all your suggestions but still lagging behind for example.. 

1. I  used Protease and Phosphotase inhibitor cocktail from CST at 1:100 concentration 

2. I normally use sample right after whole cell extraction and also use ice

3. I used 5% milk for blocking with TBS only, then used 5% BSA with TBS-T for primary antibody and finally 5% Milk with TBS-T for secondary 

4. I used HRP conjugated secondary antibodies from CST

5. Even if in very first time after cell lysis I didn't get signal

6. I tried up to 60ug of total sample but still don't get better signal 

7. I tried different blot for Phospho and total protein

So, now my question, is there anything special I need to do for transfer?

I mean, I used 80V for 120minutes transfer with 20% methanol Glycine buffer

And I used 1.0mm thick gel? 

Dear Shariful,

You might also consider eliminating Tween in the TBS.  I don't do phosphoprotein work, but I have looked for very low-abundance proteins in Western blots.  I found that for one of the proteins, Tween (unexpectedly) removed the signal for that protein (side-by-side comparison of proteins blotted to the same membrane that was then cut in half and treated the same except for using Tween).  I didn't see an increase in the background when I eliminated the Tween, but then I washed a lot for chemiluminescence since I had to do long exposures.

Good Luck!

Jill

If you are using milk as the blocking agent, Milk is heavily phosphorylated. So, you might be getting lot of background (signal to noise ratio is enormously large). Avoid milk for your blocking and antibody dilutions.. Use only BSA. 

Shariful,

      I am not sure what to tell you about your procedure if you are following my recommendations.  Your running conditions are fine for looking at phospho proteins.  

Where did you purchase your antibodies?  I am a big proponent of cell signaling technologies.  They are a great company and have great phospho antibodies.  Have you called the company and talked with the technical department?  They might have a positive control that you can run in your gels to see if at least the antibody is working properly.  

I also noticed that some people have commented that they do not use milk at all during the western process for phospho antibodies.  After multiple conversations with cell signaling and reading their data sheets for antibodies, they recommend blocking in 5% milk, incubating the phospho antibody in 5% bsa, and then using 5% milk for the secondary antibodies.  This has worked for me but I can understand using bsa for everything.

Have you also though about performing an IP experiment where you pull down sample using the total antibody, running this on a gel, and then probing for the phospho antibody?  I have done this in the past when my phospho bands were very low for certain antibodies.  

Hi Brandon,

Thanks for replying back

Yes, I used CST antibody

And now I am also thinking of doing IP

By the way do you have any better protocol for IP that I can follow?

Regards

Shariful

 Have you validated the antibodies for your cell lines or sample type? You should include a positive control for each phospho protein you're looking at. That would be a sample which is treated to induce phosphorylation of the protein. If you still don't get bands, it may be an antibody issue. Also, start blocking with 1% BSA in all incubations. If your antibodies are fresh, and your positive controls are negative, you should ask the manufacturer for a replacement product. Antibody performance can vary tremendously with cell lines and sample type.

Hi All, we work in T cell signalling, found out a best way to detect phospho proteins or any post translational modifications. As soon as the treatment is over we have to denature all the proteins as soon as possible. So we stimulate T cells or any other cells in PBS and at the end of treatment we add 2X SDS lysis buffer and lyse the cells. The cell lysate will be viscous, just do sonicaton or pass through insulin needle the viscosity will go off and this lysate use for Western. 

I agree about not using milk for transfer and using a phosphatase inhibitor cocktail. We also sometimes put the inhibitors in the SDS-Laemmli buffer (2X), heat it and solubilize the cells directly in the hot buffer. You have to leave the dyes and the beta-mercapto out of the Laemmli buffer if you want to quantify the protein levels in your samples before running the gel. You add them just before you run.

is there anyone here compared milk vs BSA for WB of p-proteins of human proteins from postmorterm tissue? I used RIPA plus all inhibitors and homogenized samples on ice with brief sonication while tube is on ice then i centrifuged in 4c and did bradford. in whole procedure proteins were on ice even while adding sample buffer then heated 10min in 70c (around 4hrs all of procedure). now i blotted a total and phospho protein and i get a clear sharp signal from total protein but only very few samples blotted for phospho form. it is like i dont see p-protein in some wells at all. the migration of protein doesnt show sign of degradation. could the effect of using milk be that much strong?

I suggest adding NaOv and NaFl to the buffers where milk and/or BSA is included, as these often contain variable amounts of contaminating PPases. Make sure to adjust the ph after adding those! Good luck

Is the Laemli Buffer appropiate for detecting phosphorylated proteins in the WB?

we go with the notion of going with bsa for all through out phosphoprotein

I have some tissue lysates I already collected some time ago but I only added protease cocktail to my RIPA buffer (no phosphatase inhibitors) as I was not planning on looking at phosphorylated targets.

Now I have some new ideas to test and would like to use these samples to do western for phosphorylated proteins, can I still use those lysates?