Yes, if the antibody is stored for a long time under non appropriate conditions.

If the antibody is denatured or partially denatured, you will have hydrophobic interactions which are unspecific.

I agree with Michael's answer.

as a last trial,

you could try to spin down (10000g*10min) your Ab vial,

recover the supernatant and add some bsa (1-3%).

the day after (e.g.), spin down and take your Ab for dilution.

if some specific staining is left, you should see it.

Antibodies have the property to aggregate when they are purified. So you can have different results the time.

1. Therefore use aliquots which haven't to used for longer periods. Sometimes it's helpful to add some inert proteins like BSA. This keeps the water also around the antibody molecules. Aggregates will increase the background and so you will have a minimized signal/noise ratio.

2. If you store antibodies frozen, store them below the eutectic point (-25 °C).

3. In rare cases you have an bad preparation which contains also some proteases. Here also an inert protein can be helpful as well as a protease inhibitor (or deep freezing). The only problem, there is no inhibitor for all kinds of proteases but PMSF (1 mmol/L) works in most cases.

4. Proteases can reach the product as well by bacterial contaminations. NaN3 is the best choice.

Thanks to all,

The Ab I m using is a chimeric Ab where a protein is fused with the Fc part. for this reason I perform a blocking step using a FcRg blocker that should block all the Fc binding. Also the cell line I tested is NIH3T3 that is fibroblastic line. I will try to redu ce

the concentration of the Ab and I will increase the % of FBS in the buffer solution.

How many times should I wash the cells after the staining and how long should I incubate the Fc blocker?

Thanks

Incubate cells with Fc-blocker for 15 min then stain without removing Fc-blocker.

2 times washing is usually enough.