Do you have something to run as positive control, such as recombinant proteins? The primary antibodies you are using, how they have performed in previous reports? I could not understand the attached image? The top panel is your sample but what shows in bottom panel? Product of some housekeeping gene?

Yes Sir, the bottom panel is beta actin from same samples.

Dear Mansi 

It seems that the expression of NOS2 is low to get around this you could try the following: 1) Load more protein like 50ug if possible, 2) use a positive control to stimulate expression of NOS2 e.g. Clostridium botulinum toxin C3 ribosyl transferase (C3) (treat with 63ng/ml for 24H) or 3) leave your membranes for 48hr in primary antibody (1:1000 TBS-Tw or 5% milk) shaking at 4oC then perform secondary incubation as you normally do (I found this to help detect low abundance proteins)

Good luck with your experiments and do let us know if you are successful in detecting NOS2.

Thanks and regards

Luke  

Hi Mansi,

What I would do is to use a positive control, as Syed and Luke suggested, but then incubate with your primary over the weekend and use more of your secondary antibody (like 1:4000, or even 1:2000).  This way, you can make sure you're seeing your band of interest.  However, this won't solve the background problem.  In fact, it will probably make it worse.  So, what you can do is, once you've ensured you're seeing the right band, optimize the amount of time that you are blocking (I usually do 90 minutes to 120 minutes @ RT with 5% NFDM).  That way, you can make your signal look pretty.

One other thing to consider, too, is the amount of time for your transfer.  Whenever I investigate high molecular weight proteins, I often have to extend the amount of time that I transfer by 10-20% (e.g. I usually transfer an hour for 60-80 kDA proteins, so I usually go 75-80 minutes for 150kDA or higher protein).  This may help cut down on your background and improve your signal.

Anyway, good luck in your experiments.

Cheers,

Bryan

Your antibody conditions might be incorrect, or they are not specific enough for NOS. Multiple banding is often the case with poor batch quality or polyclonal antibodies (primary or secondary). You can check the specificity of the secondary antibody by treating the membrane with only the secondary and then developing, and seeing what bands appear. Any banding is non-specific binding by the secondary to other than the primary antibody directed at NOS.

Good luck

Dear Bryan Sir,

Thanks for the informative suggestions, please suggest me the transfer method, should it be done at constant voltage(40-50 Volt) or constant current (20 mAmp), I have tried overnight transfer considering transfer of high molecular weight proteins. Will overnight transfer make a difference?

Hi Mansi,

I usually use constant voltage at 100 Volts, but I am using miniprotean gels and nitrocellulose membranes.  If you're using PDVF membranes or a different size gel, it might be completely different conditions that are optimum.  You can try doing it overnight, but it will probably increase your background.  That's all I can say without knowing more.

Jeremy has a good point about the antibody that I didn't think about.  You might need to "shop around" for a better NOS primary antibody.  However, primary antibodies are pricey, so, if it were me in your situation, I would try to optimize your conditions, as I suggested in the first post.  But at a certain point, saving time becomes more important than saving money.  Only you and your lab know what that point is.

Anyway, good luck and I hope I've been helpful.

-Bryan

Dear Mansi,

It also depends whether you are using semi-dry or wet transfer. Generally speaking, people seem to prefer wet transfer for large proteins and semi-dry for small ones. In our lab, we also have better results using Nitrocellulose membranes and transfer buffer containing SDS for high molecular weight proteins.

Good luck!

Sina

nice explanation Bryan B. Bridgeman  sir

Hey Mansi,

                 Especially when protein expression is very low, try to run freshly prepared samples and load as much as possible. Try incubating membrane @ 4 C for overnight (18-20 rpm shaking) with primary antibody.  (also try incubating membrane with antibody at RT for ~2 Hours). I would suggest You to check pH of TBS which should be ideally 7.5. After secondary antibody incubation wash the membrane thrice with TBST vigorously that would remove your background signals. 

All the best!

Thanks Kishore

You can optimize transfer conditions by trying a few combinations of voltage and time with a set of pre-stained protein molecular weight markers that has at least one protein that is larger than the mol wt of your protein of interest.