Hi,

The blocking step is very important  as it will reduce the un-specific bond of your primary/secondary Abs to the membrane (they are proteins, after all). I used to block the membrane for at least one hour in 5% milk solution (or blocking buffer if infrared imaging is used) and incubate the membrane with the primary antibody over night in cold room, shacking (increase specificity). Regarding the secondary, 1-2 hours at RT would be sufficient.  Washes (3 x 10 min each, for each step). Usually this protocol work very well.

Stripping is an harsh procedure that may affect the quality of following WB. I used to block again before starting with the primary. I used to skip stripping if I can. You may not get decent results and I prefer to run another gel.

Why stripping for blotting Loading Control? Can you cut the membrane in 2 pieces and blot for your protein of interest and LC in two separate boxes,? If you cannot because both are close, try to run the gel longer or play with the % of your gel for a better separation. Increasing the primary Ab/reducing secondary Ab concentration (get higher specific signal during ECL will  reduce the exposure time of your films), may help?

Hope these suggestions will be useful. Good luck.

Regards,

Daniele

Hi Daniele,

Thank you for your suggestions they are very useful and I will give them a go. Just wondering though, why the same background signal would persist after stripping and incubating in antibodies in blocking buffer? Wouldn't you expect that background signal to change? Or do you think this means that the stripping/ blocking did not work very well?

Many thanks,

Rachael

Hi,

I would expect the background will increase after stripping. Usually it happens. Some primary antibodies are specific but generate background by default.  As general rule, monoclonals are better than polyclonals (not ever...).

Regards,

Daniele

Daniele is correct, you will definitely want to block your samples prior to applying primary antibody in order to reduce the background. This is still necessary after stripping because doing so should remove all binding, not just that of your protein of interest.

As for blocking itself, many people use the 5% milk block suggested by Daniele, but if you're willing to spend a little more money, many companies have developed proprietary blocking agents that help reduce background. I have had very good luck with GE Healthcare's Amersham ECL Prime Block (product #RPN418) at 2% in TBS-T. It may be worth giving a try if you're not getting a good signal with milk. Good luck

My main problem sets to be that the stripping removes most of the specific staining but leaves most of the nonspecific staining. Whereas I would have thought the non-specific interactions would be weaker. 

Thanks for your comments.

I guess you would want to try and reduce the background on the first blot so that it won't persist to your second block. Have you tried adding tween-20 or triton x-100 in your primary antibody buffer? It may help to reduce any non-specific binding. 

Otherwise, as mentioned above - you could always cut the membrane and do both blots simultaneously in separate boxes. 

Hello Rachel!

It happened to me something simmilar to what happens to you. I think your problem is the TBST buffer. It does not have the proper pH.

My protocol to do TBST is:

8.8 g/L NaCl

0.2 g/L KCl

3 g/L Tris-Base 

pH=7.4

after that you must add 1 mL/L of Tween 20

Best wishes with your experiments

Francisco Llavero

Thank you very much. That makes sense that the washing isn't stringent enough.

If you are using the same secondary for both blots you might see the same non-specific bands.

All the strategies suggested by other contributors are the logic way to get rid of your background but also consider this...

In my experience secondary antibodies are the one that enhance the background provided by the primary Ab. First, be sure that you antibody conjugated to HRP is not degraded or very old. You can also increase dilution (make it more diluted) for your secondary antibody to decrease background (you can increase time of incubation).  Reblotting is always difficult, maybe you should try different primary antibody as well, monoclonals give cleaner bands. 

My biggest concer is about your "expected" bands going away and the "non-expected" remaining, this also suggest that you have a promiscuos Ab.  Again, you should try a different brand, origin (species) and dilution. 

Be patient, you will make it

I might be able to help if you provided detailed information about your protocol.  Ex.  Membrane type, solution composition, incubation times, etc... Sources of background are caused by different things when using different detection methods.

Try Sea Block Blocking Buffer, Thermo Scientific, catalog #37527.  Good Luck, Ron

It is not "non-expected" bands that remain but rather the background noise all over the membrane

The SeaBlock should help with that.

If you have very high background to begin with, it means that you need to optimize your blocking buffer and ensure your wash solutions and incubations are good enough before working on stripping. For instance, we found that adding 0.05% Tween in the blocking buffer helps in increasing the signal to noise ratio of the blot, but all these factors need individual optimizations for the blotting system you use (membranes, etc). Your widespread persistent background might indicate issues with a more basic buffer like the wash solution whatever that is (PBS or TBS). Also you might need to check the pH of the buffers. 

Sometimes it nees a bit of a twist. I currently use 10% skim milk in 0.05% PBS-T as our blocking solution. Our background is really good. I compared a few blocking solution (non Tween-20; 10% vs 5%; BSA vs milk0, and found the one that work for us. so you can optimise to find the one that work for your equipment there as well.

Good luck.

Hi Rachael,

We normally wash the membrane after exposure with PBST. Strip for 30-60 min, wash extensively afterwards, and block the membrane for at least 30 min before probing with your new antibody. This helps with the background.