have you tested already different blocking reagents?

BSA, milk ... in different concentrations?

you should also add BSA or milk to your primary and secondary antibody.

maybe you also have to block for a longer time

is the antibody specific? Do you know that you should just see one band?

all the best,

Sebastian

The measures that are important to be taken is Increase the washing step after each antibody and also increase the blocking time.

Use BSA. This is basic comment, but wash thoroughly after each antibody incubation. You will get better at this.

I do always 3x10-15minutes washing after each step.

I think you can never wash too much ;)

Proper Antibody dilution and proper washing solves the problem.

Other than longer blocks and using milk/BSA in your antibody solutions, if you have strong specific signal, try a lower dilution of secondary antibody. Peroxidase conjugated antibodies can be diluted up to 1:5000 or more and would still give strong specific signal with lot less background.

As others have said really, I find a 5% milk solution in PBS and block for a couple of hours, then incubating the antibody in the milk solution helps. I would also wash at least 3 times for at least 15 mins after primary and secondary anti body incubation. Also how strong is your band? If it is very strong you can expose the film (assuming you are using x ray film) for less time.

Thank you all.

I tried changing the wash time and also tried a varying antibody concentration . I have been doing westerns for a pretty long time, and suddenly I get so much of background now. Is there a possibility of the secondary antibodies have become older. I did not find any problem with mouse monoclonal , but only with rabbit polyclonal antibodies. Is this has to do with the background.?

Some suggestions without knowing the details of your protocol:

1. Is your membrane glowing in the dark? If so, dilute primary/secondary antibodies more (as suggested), or even dilute the developing reagent.

2. High background can be the result of too much developer on the blot. Take a kim wipe and literally dry all the excess off.

3. As mentioned before, wash more. After primary or secondary, I actually rinse the membrane with DI water first, then do 4 washes in TBS-T for 5-10 minutes.

I have found 5% milk works best in this situation, although it is very antibosy dependent.

Also happens for older blots that have been stripped and resued

I'd go for diluting the sec.AB and increasing washing as mentioned above.

However, If that does not work you might want to look into your washing buffers.

Add autoclaved detergent to your buffer freshly every time. Fungi can grow in buffers with detergent in them. Using "old" detergent might contribute to background due to the mess the fungi makes.

It is a more exotic problem admittedly, but might just do the trick. Best of luck.

I may have missed it, but what type of membrane are you using? Nitrocellulose? PVDF? I very rarely get background on Nitrocellulose. But on PVDF I have to make sure to was at least 3x for 15 min each in TBST. I've found having the T (tween-20) in your TBST is very important in helping to reduce the background.

Also, as suggested above multiple times, having fresh 5% milk-TBST for blocking is pertinent; using secondary (or primary) antibody that has gone off will also ruin your day.

Best of luck!

As you said regarding you mouse secondary Ab, did you try using a fresh stock? (may be from neighbour labs). It is also worth to note that the protesae inhibitor might not be working(I hope it is there in the lysate)? Check whether the Ab raised against IgG or IgM, sometimes it really really matter!

If your protein of interest is also abundant try diluting the start material.

Good luck!

You must to optimize the concentration of primary and secondary antibodies as well as incubation time. I recommend you: protein blotting guide. A guide to transfer and detection, 3rd edition. Biorad.

Possible causes:

1. Blocking was incomplete

2. Insufficient wash protocols were used

3. The blot was left in the substrate too long

4. Contamination ocurred during electrophoresis or transfer

5. Excessive amounts of proteins were loaded on the gel, or too much sds was used in the transfer buffer

6. The primary or secondary antibody was too concentrated

7. The incubation trays were contaminated

regards

Try SuperBlock from Pierce

Thanks all for your help. Il try all the possibilities you have suggested.

The problem in wash

try 3 times after 1st ab (each time 10 min),,, after 2nd ab 3 times each time (10 min)

I hope you FILTER the final DEVELOPER solution. Because precipitates will give such background