It's possible that a harsh stripping removed your protein to a certain extent making it hard to redetect HDAC1. Increasing loaded protein amount, antibody incubation time or improving your transfer could help getting a nice detection. How are you performing your immunodetection?

I used 45 ng of protein for loading and have done overnight transfer and stripping was done at 55 degree for only 15 mins and primary antibody was shaken at full speed for 1 hr and then was put at 4 degree overnight. next day it was again shaken for half an hour.secondary antibody was 1: 5000 and for 1 hour.. Antibodies are 4-5 years old , but was thawed only for few times. does this could be a reason for no signal

Hello Lalita

I completely agree with the last speaker. Stripping is designed to do just that and can, if too aggressive, denature/strip away your protein/epitope and consequently reduce specific signal

Did you strip at 60oC ? If so try incubating with stripping buffer at room temp for 10-20 min. As already suggested increased lysate loading will compensate for any loss incurred by stripping.

I did stripping at 55 degree for 15 mins

Hello Lalita

sometimes stripping at lower temp can be less aggressive but less effective

45ng of lysate is definitely at the upper end of how much you should add so I wouldnt recommend adding anymore; I typically add 10ng to 30ng

You did say that after 1 hour you only got a faint signal before stripping so i Suspect the main problem might be your antibody: either the age of your antibody if already validated or even a new antibody can on occasions not performed as expected. Is this antibody known to give a specific signal ? Either people you know or at the very least verified in independent publications. I have for example tried to Western blot various proteins and been obliged to use 2 or 3 different antibodies before getting a specific result

On the subject of stripping I tend to strip having performed specific western blotting in order to re probe with a housekeeping antibody like b actin or GapDH to verify uniform loading. If that doesnt work then you can be sure you have problems with aggressive stripping, quite aprat from the age and/or specificity/avidity of your primary antibody

Hi, did you run any control with your HDAC1 western? I am not sure how good is your antibody but I usually load 10ug of total protein lysate per well, depending on your expression. As for stripping control you can do the same western for GAPDH or any housekeeping protein to see if its harsh or not.

Hii Lalita, as per my experience, use fresh membrane for immuno detection as far as possible. Stripping always leads to loss of some amount of protein from the membrane. Since, you were able to get faint band at 1:1000 dilution of primary Ab, increasing the incubation time may help you (incubate overnight at 4 degree C at same dilution). Also you may incubate for 1-2 hours with 1:500 or lesser dilution of primary Ab (You have mentioned your antibody is years old. so that might have reduced its efficiency). All the best

Why don't you try different concentrations of your protein together with a positive control? let's say 20, 40, 80 ug of protein lysate. May be your gene of interest is not highly expressed. Also it is not recommend to strip and reblot the same membrane with the same antibody  when you don't  have standardized conditions.

HDAC1 is  a nuclear protein, prepare nuclear lysate and do Western or else lyse your cells in 1X SDS loading dye this will lyse the cytoplasm and nucleus, upon lysis cells will become viscous, use sonicator to decrease viscosity. If you use triton X lysis buffer this will not open the nucleus, NP40 and RIPA are also good for breaking nucleus. 

Hi, I completely agree with Anbalagan, and I think your protein extraction method has some problem... And use some postive control if possible.

And follow the above method for your protein extraction.

Best

Hi, I am working with same antibody (anti-human HDAC1 from Cell Signaling Technology). It is working fine within my experimental conditions. I am loading 10 ug of cell lysate (prepared using Tris-NP40-EDTA lysis buffer), blocking with 5% milk powder for 2 hours at room temperature, primary antibody (1:1000 in 5% BSA solution) incubation kept it for minimum overnight on roter/shaker (not too strong, just to ensure that antibody solution is running over the blot) and then secondary HRP-conjugated antibody (1:7500) for minimum 1 hour and develop it with ECL solution.

Best luck!

That sounds like qa standard ECL WB protocol that I have successfully used with quite a number of antibodies so fully endorse what the last participant has written :-)

Thank you

 I also use the same lysis buffer and do blocking for 1 hr. primary antibody 1:1000 , Full speed shaking at RT for 1 hr. and then overnight incubation at 4 degree without any shaking[ where do you put your memb. with overnight shaking(4 degree or RT) as overnight at RT has risk for protein degradatin, so cannot leave at RT overnight] , then secondary 1:10000 n tried 1:5000 also) for 1 hr.and develope with ECL, so where is the fault? I am getting only faint bands. could it be because of 4 year old antibodi

1) yes, it's quite possible that faint band is because of old antibody. Usually if antibodies are stored at recommended condition, it should not be problem. 2) If you want to test your antibody, you can try to use other cell lines like HeLa, COS, showing positive expression to compare. 3) You mentioned, 'blocking for 1 hr. primary antibody 1:1000 , Full speed shaking at RT for 1 hr. and then overnight incubation at 4 degree without any shaking'... sorry, may be its not clear yet, so again my protocol --> Transblot --> 5% milk powder blocking at RT for 2 hrs, shaking (not too strong), just to cover up blot with solution --> wash with TBST at RT for 10 mins each, 3 times, shaking (not too strong), just to remove residual solution --> primary Ab in 5% BSA, overnight at 4 degree, slowly rotating, just to cover up blot with Ab solution --> Wash with TBST at RT for 10 mins each, 3 times, shaking (not too strong), just to remove residual solution --> 2 Ab (HRP-conjugated) --> Wash with TBST at RT for 10 mins each, 3 times, shaking (not too strong), just to remove residual solution --> fresh ECL solution 1-2 mins --> Develop.

Best luck!

Thank you so much for this elaborated protocol and a valuable suggestion. I will follow the same and hope this time will get band . we stored the antibody in - 20 degree since then and it was thawed only few times. The protocol I mentioned was the protocol  after which I got that faint signal. Now I will follow the exact protocol as of yours and yes , the cell line also matters, may be my cell line (DU145) expresses it at lower level.That's why I am getting the faint band.