I have had it quite rough with pCREB. I think its too fragile and really needs you to be quick when extracting the protein and mantain low temperatures as much as possible at every step. And make sure you have phosphatase inhibitor in your lysis buffer. The antibody (p-Ser 133) binding is very specific, so needs longer time (overnight, 4C!)

Also, I have just noticed it is better to add the loading buffer to the sample soon after extraction if you are going to store it for later use. I used to extract and freeze to -80 for later use and then add loading buffer just before loading for SDS-PAGE, but I seldomly got the desired bands!

I will be glad to see more tips here too! Am still working with pCREB and CREB western blotting.

The results of WB usually are presented as spotted membrane image. For accuracy index of procedure, at list 3 membranes of 5 repeats, must show the same results. the extracted data from these membrane are usually ideal for acceptable error bars. higher reproducibility is the main factor.

The methods for reliably visualizing proteins via Western blot are many, and depend on the technique and skill of the person performing the technique. Therefore, the generic answer is that the most reliable method is the one you find works best and most frequently. Limitations also depend on the detection methods available to you.

I agree with Dr. Habibian concerning N number for acceptable error bars. You optimally need at least 5 samples (one from an individual animal), and their blotting result should be repeatable times. Of course, the more samples you have the better, and be sure to optimize the method by which you are performing densitometry or quantifying the protein band(s) of interest. Although you may meet the N and replication requirements, your quantification method can lead to unrepresentative variation in your results.

Thanks Reza and Chandler for your answers. Do you have any experience with pCREB western blot?

I am actually working on that right now! I haven't finished the experiments, but I will try and post on here and tips or problems to look out for.

I have had it quite rough with pCREB. I think its too fragile and really needs you to be quick when extracting the protein and mantain low temperatures as much as possible at every step. And make sure you have phosphatase inhibitor in your lysis buffer. The antibody (p-Ser 133) binding is very specific, so needs longer time (overnight, 4C!)

Also, I have just noticed it is better to add the loading buffer to the sample soon after extraction if you are going to store it for later use. I used to extract and freeze to -80 for later use and then add loading buffer just before loading for SDS-PAGE, but I seldomly got the desired bands!

I will be glad to see more tips here too! Am still working with pCREB and CREB western blotting.

Thank you for the information, Stephen. I will keep these tips in mind.

Thanks Stephan. Yes working with pCREB is tricky especially if you want to quantify the results and if the changes are low. which buffer did you use? RIPA? I used RIPA fist then I changed my method to boiling the sample directly into sample buffer which was very good in the beginning but the result was not the same as first method.(still donna why and which is the correct one!). Do you use Tris Glycine gel or Bis Tris?

Thanks again

Pooneh, I use RIPA buffer and Tris glycine gel. - I have never tried the direct boiling method. I'll probably give it a try some day & see how it goes!

Dear Pooneh and Stephen, I see that my former hints on pCREB Western blots were not helpful. What I can offer both of you is to send me your samples (control and treated) preferentially extracted with the direct sample buffer addition method or whatever you use instead and I do the rest in my lab and send you the results. As author of several papers with pCREB Western I feel bad about the problems you have. I think it has to do with lower quality of recent commercial S133-phosphorylated CREB (pCREB) antibody preparations. Maybe I can help you fixing it. Best wishes, Erik

Erik,

Can you provide some examples of commercial suppliers who are selling quality p-CREB s-133 at this time? I know different vendors have different reputations of primary antibody quality, but this can vary from antibody to antibody. Thanks.

Dear Erik, your points were very useful. the problem I had with boiling is 1- I could not reproduce my previous results using RIPA and phoshphatase inh. with boiling method and 2- I got some smear bands and it was not predictable because I prepared all samples the same way but unfortunately some were smear. (maybe degraded). So for my new samples I am back to RIPA method. I think the antibody is not a big problem because I get the bands although not very strong the problem in my case is I do not know how reliable is the result. I am afraid that sample preparation affect the result more than the real difference. I am working on that now. Thanks

Thanks Erik for your kind offer but I think it is not possible at this time. BTW I really appreciate.

My pleasure Pooneh!

May I ask if you used SDS- or LDS 2x sample buffer and which temperature did you use? LDS based samples should not be hotter than 70 degrees Celsius.

To Walker and all others interested. These days in my hands only Cell Signaling #9198 phospho CREB (Ser 133) (87G3) rabbit monoclonal works acceptable for Western in cAMP analog-treated (Sp-cAMPS) or forskolin-treated HT22 cells, a cell line from mouse hippocampus. This Ab also works in forskolin-treated rat pinealocytes (one hour stimulation all). It does not work in immunhistochemistry or immunofluorescence applications on cells or mouse hippocampal slices. The Millipore (formerly Upstate) pCREB which worked also nicely in the old days does not do so anymore.

Thats all I can say for the cuurent situation, however I have ordered some more pCREB Abs but have not tested them so far. These days I have a lot of teaching to accomplish, news from my side on this issue are to expected not before the end of February 2013.

Erik, that's so kind and helpful of you- Thanks for the offer... Am already getting there- almost done with my results (I guess am fine now & may not need to send my samples, but thanx anyway). Your earlier comments have been helpful beyond doubt, and my success with pCREB upto now can partly be attributed to your advice!! (I, at some point, had to exchange an antibody for another batch from the same company, and used new phosphatase inhibitor).

New comments and tips are always helpful and appreciated.