Hello Dr. Schmitt, Thanks for your suggestion , yes i m using protease inhibitor cocktail from sigma, and it recognize my desired protein along with some other. i have not check yet in knockout gene sir, i was just doing with my treated sample and got those bands. looking forward to hear from you soon.

Thanks

A negative control would be helpful for troubleshooting. You could either use cells from a knockout most or a tissue that you know does not express the protein that you are trying to detect.

Try reducing the amount of primary Ab and then the secondary Ab. Use large volume washes when you are washing the blot.

If this does not work, try a different primary antibody.

Hi, Dr. Owen thanks for your valuable suggestion i will go do as you said and will see the results and will take the negative control for better troubleshoot purpose.

thanks again.

I would add following things: 1) How much % of tween you have added in TBST that also matters, try 0.1% tween containing TBS; 2) Blocking buffer needs to prepared in 0.1% TBST; 3) Don't wash after blocking; 4) If I use 3% BSA for blocking then I prepare my primary Ab in 1% BSA and not just in TBS or TBST (BSA containing buffer helps in sustaining the Ab for longer time at 4 degrees). I don't prefer to use skimmed milk for diluting the antibody only because one can use that Ab solution only once or hardly twice (not advisable as milk can not sustain for longer time). I know that it is not best to reuse the Ab but due to the reason that some of the Abs are pretty expensive, I use the Ab diluted with BSA at least 3 times (depending upon the protein). 5) I use the same buffer for diluting the secondary Ab that I use for my primary that is 1% BSA in 0.1% TBST and incubate for 1-2 hours at RT. 6) You also need to optimize your ECL's incubation time. All the best!

Hi Dr. Divya Bhatia , Thanks for discussing so many things about this and yes i am taking 0.1% tween20 in 1X TBS. Generally i wash my membrane after block as i ma making the blocking solution in 5% milk let me try this time in BSA .yes i use TBST for AB dilution not BSA same for the 2ndry AB let me change those things and will see the results.

thanks.

If you don't have any KD or KO negative control, you could try to decrease the concentration of your primary ab to 1/2000, or using 2.5% skim milk for both primary and secondary Abs (if skim milk is better for your blocking). If you are looking at a phosphor protein, it will be better to use 5%BSA for blocking and same buffer for Ab dilution. If you want to reuse you Ab, you can consider to add 0.05% Sodium Azide to your final Ab solution and keep in 4 degree. I can reuse my Ab with 0.05% Sodium Azide for more than 3 months in 2.5% skim milk in TBST. Good luck with your WB.

Hi!

1.blocking with 5% skimmed milk for 3h followed by washing with 1X TBST thrice.

Use 5% skimmed milk dissolved in TBST 0,1%. - wash five times for 5 minutes with TBST 0,1% (tween).

2. primary Ab was taken with 1: 1000 for 17h treatment follwed by washing with TBST.

Try dilute your Ab to 1:5000 and/or incubete for 2 hours. Wash 5 times with TBST for 5 minutes with agitation.

3. 2ndry Ab was taken 1:8000 for 3h followed by 3 times washing with TBST

and then developed by using ECL solution in x-ray film

but still i am unable get the target results. please suggest me how can i get rid off this problem

Wash 5 times with TBST with agitation.

Best,

Jacó

Hi Jacó Joaquim Mattos , still i am doing the troubleshoot, as per my experience i suggest go for more dilution of the secondary AB, after i did i am getting better results then before.