starting from the basics: do appropriate blocking (5% milk in TBST or 3% BSA in TBST) for no less than 1-2h at root temperature. whashing steps: 30 min each, at least 3 washing steps after the primary and as many after the secondary.

Impossible to know without a representative image. Can you post one please?

I do not have it now on software

i will scan the film and post

thanks alot

starting from the basics: do appropriate blocking (5% milk in TBST or 3% BSA in TBST) for no less than 1-2h at root temperature. whashing steps: 30 min each, at least 3 washing steps after the primary and as many after the secondary.

Hi, Shahinaz. How specfici your antibody?

Do ypu mean low signal/noise ratio?

Do you have only background or some non-specfic bands?

I think you should try to reduce concentartion of the primary antibody, reduce temperature of the incubation (4°C, overnight, for exmaple), add 0.1 or even 0.3% Triton during washing.

And, finally, I would suggest you to not shake (aginate) membrane during antibody incubation.

Good luck!

Is it only the background that is bothering you or you are also getting some non-specific bands? If the issue is only with the background, then I must say that blocking must be done well (overnight blocking with 5% milk in TBST). Also, apply both primary and secondary antibody (in blocking buffer). Washing at each step must be done with TBST without the last two step before developing.

Use non fat dry milk as blocking agent 2-5%

Thanks very much to all of you for your precious replies,i will try taking care of all you suggestions in my next western

Dr. Taras Pasternak

Thanks very much

i am concerned about both background and non specific bands

my last blot was B-Actin

it gave me very dark image and bands hardly seen inside this black film

Dr. Pritha Dasgupta

Thanks for reply

Yes i have both issues

i will fellow your directions

hoping to get better results

Dr. Francesco Roselli

Thanks very much

i do wash for only 15 minutes but still 3 times

well,i will double the washing time

Others have listed good general suggestions (blocking buffer composition, wash buffer composition, antibody dilution buffer composition, etc). However, without knowing a number of experimental variables (I.e. sample type, loading concentration, detection method, lysis buffer composition, antibody isotype, transfer buffer and method, nitrocellulose vs PVDF, etc), it will be difficult to pinpoint one or two specific issues. A representative blot and procedural detail will help immensely with an accurate answer!

Does the background have many dark spots. That means you may have clumping of blocking agent. Google for Western blot troubleshooting. There are answers to each of the problems you may face with Western blots or any other procedure

Here is a good source

http://www.abcam.com/index.html?pageconfig=resource&rid=11352

If you are sure your sample is well enough prepared and there are no clumps etc then try to run the gel slowly and completely. Block overnight at 4C if you have a lot of background. Wash diligently at appropriate times. 3 times are sufficient but they need to be done diligently. Try using less primary or secondary antibody to understand which binds elsewhere and gives background. Generally speaking gentle shaking gives better results. Not rushing the procedure will give cleaner results from the protein production, through gel running and staining if the problem lies with the actual procedure. If others have used the same antibody in your lab or elsewhere, ask to find out whether this particular antibody set has given them trouble. Perhaps they faced the same problem and troubleshot it. Good luck!

Inefficient washing during primary and secondary antibody incubation may be the reason for darker background. Other reason might be over incubation with secondary antibody for longer time. Wash membrane for 10minutes 3 times during primary and secondary incubation. Use secondary antibody dilution (1:10000) for 1 hour at room temperature. Try to use BSA (5%) as blocking agent) and dilute your antibody in 1% BSA. Which membrane you use for western transfer PVDF or nitrocellulose?

Dr.Sandeep Rajput,

thanks for reply

i use PVDF membrane

i block with 5% non fat dry milk on PBST 1X for 1 hour @room temprature

2ry antibody dilution 1:5000

incubate 2ry for 1.5 hour @RT

Next time try 1:10000 dilution and incubated for 1 hour at room temperature and wash ur membrane well each time. It will work for sure.

Do not block for more than one hour. Immediately after blocking give a small wash with TBST before adding primary ab of interest. Ensure that you haveadded twin20 in TBS buffer for washes. After probing with primary and secondary give four 15 minute washes. Then you will definitely get good results. All the best.

Maybe you should also try to reduce the secondary antibody concentration and/or try a different blocking as suggested by Franscesco. Good luck ;)

Assuming that you are not using too much antibody as suggested by others, another possibility is that you are waiting too long for the blot to develop so that you can detect the bands. In that case, I would increase sample load. If you have the wells loaded to the maximum volume, try using 6X sample loading buffer so you can add more sample volume, or concentrating the sample.

Eric A F Herbst thanks very much

Sure,this is very helpfull

In addition to the other suggestions - the best chemiluminescent detection reagent on the market when using an HRP-linked secondary antibody is Super Signal West Pico, which is sold through fisher.com - it has great sensitivity and very little background when compared to a similar product from Millipore - using a very, very sensitive reagent like the one from Millipore is great if you are trying to detect faint bands, but the background will be higher because it's super sensitive. The other thing to think about is your primary antibody reagent. If you are using an anti-his antibody, there are many on the market and some are not very good and cross react greatly. The most specific anti-his antibody is from Genscript and is part of The Elite Antibody series. You can get a free 10 ug sample to try this antibody if you are trying to detect a his tagged protein - here's a link (http://www.genscript.com/THE_antibody.html) - if your primary antibody isn't very specific, it won't matter how much you block, wash, etc., you will see cross-reactive bands. I'm assuming that when you say your background is high, you mean that the blot is dark, so it's hard to see the bands. This can usually be optimized by focusing on the blocking step, or the exposure time when you add the film (less exposure, maybe one minute instead of 3 minutes, for example), may help to reduce the high background. In addition to blocking reagent, the detection reagent makes a difference, as does the quality of the primary antibody (specificity).

can rinsing with distilled water for 1-2 minute before developing, remove the nonspecific bands?

Blocking the non-specific proteins with the blocking buffer would remove the background signals. Make sure you use the right formulation. 3%BSA in TBST or PBST should be OK. Another means to reduce background signals is your washing process. Dont forget to wash with PBST or TBST 3 times (5 minutes each after adding both primary and secondary antibody). Some protocol may say it is good to wash once after transfer, I dont wash after transfer, I block immediately and I have good background.

backgroud is usually due to lack of washing. You can wash with PBST for 30 minutes 3 times ( put on shaker machine ) . in some cases , blocking agent BSA better than skim milk

 Thanks so much Adita, i appreciate your help

High background across blot is a common issue. Most of the time it is caused by insufficient blocking of nonspecific sites. You may want to increase the concentration of the blocking reagent. I usually use 3% BSA and it works fine for me. If you are PVDF it is important to add 0.1% Tween-20 to your TBST buffer (assuming that is what you use).  Furthermore you may want to increase the number of washes and the volume of buffer you use. 5 washes for 5 minutes in 10 ml buffer work well. Avoid dried membrane during the blotting procedure can also be beneficial in reducing the background. Good luck.

Appropriate Blocking is the key.

There could be numerous reasons for the high background such as:

1. Blocking of non-specific binding might be absent or insufficient

Increase the blocking incubation period and consider changing the blocking agent, we recommend 3–5% non-fat dry milk, BSA, or normal serum for 1 h at room temperature. These can be included in the antibody buffers as well.

2. The primary antibody concentration may be too high

Titrate the antibody to the optimal concentration. Incubate for longer but in more dilute antibody (a slow but targeted binding is best).

3. The incubation temperature may be too high

Incubate membrane at 4°C.

4. The secondary antibody may be binding non-specifically or reacting with the blocking reagent

Run a secondary control without the primary antibody.

5. Cross-reactivity between the blocking agent and primary or secondary antibody

Add a mild detergent such as Tween 20 to the incubation and washing buffer.

For phospho-specific antibodies: milk contains casein which is a phosphor-protein; the phospho-specific antibody will detect casein present in the milk causing high background. Use BSA as a blocking reagent instead of milk.

6. The washing of unbound antibodies may be insufficient

Increase the number and time of washes.

7. Your choice of membrane may give high background

Nitrocellulose membranes are considered to give less background than PVDF.

8. The membrane night have dried out.

Care should be taken to prevent the membrane from drying out during incubation.

For more troubleshooting details, you can refer the following weblink.

http://www.isogen-lifescience.com/blotting/western-blotting-documentation/high-background-in-western-blots---causes-and-solutions

I would have a couple of suggestions that you could also consider:

• Protein abundance: in case the target protein abundance is lower than the threshold of nonspecific binding you might need to load more protein per well at SDS-PAGE. PaxDb: Protein Abundance Database could be useful here: https://pax-db.org/ If you do not have enough of your sample try to enrich low-abundance proteins by immunoprecipitation.
• Samples degradation: if only possible, always work with a fresh lysate. I would also suggest sonication step. In case you are working with cells, please sonicate for 1 min and 2-5 min for tissue samples (180 watts; 2 sec ON/2 sec OFF).
• β- Mercaptoethanol and DTT reducing agents: you could also try using 20% β- Mercaptoethanol (or 100 mM DTT) for the 4X SDS sample buffer that could help reduce the background staining.
• Importance of blocking step: you might also consider blocking step overnight, especially while working with tissue samples. Various blocking buffers are available, and not all of them work in every situation. Keep in mind that milk-based blocking buffers contain endogenous biotin, glycoproteins, and enzymes that may interfere with the signal. Milk-based buffers also include active phosphatases that will sabotage your detection of phosphorylated proteins; in these cases, try alternatives such as bovine serum albumin-based blockers.

Find more tips here: WB high background troubleshooting https://www.ptglab.com/support/western-blot-protocol/western-blot-troubleshooting-high-background/