Hi Himangshi,

There is no straight forward answer available to your question. Primary antibody dilution has to be standardized individually depending upon the species cross reactivity (immuno affinity) and actual stock antibody concentration. Monoclonal antibodies work better at high dilution compared to the polyclonal as well. Odyssey Li-Cor has a device that is widely used to determine antibody titer per channel (well in WB) using varied concentration of antibodies of interest.

The easiest way to overcome your problem is to begin with a higher dilution (may be 1:3000 or 1ug/ml you have to determine) for a longer duration of incubation at 4 deg C and you can play with your secondary antibody (increased concentration/ increased incubation time) as well. I am not sure what detection method you are using: Chemiluminiscence or IR?

If the higher primary dilution does not work then increase the concentration in a smaller increment to the same diluted antibody without throwing it away. I usually reuse the primary antibody dilution if kept carefully at refrigerator (4 deg C) without any contamination at least for a month.

To minimize the working volume of diluted antibody you can roll your NCP or PVDF into a 15 or 50 ml Falcon tube and add 2.5-3.0ml antibody dilution in the blocking buffer and use a roller mixer for incubation.

I hope these ideas work for you.

Antibody dilution depends on the affinity of your antibody. It can vary from 1:500 to 1:5000. You are the best person to know which dilution works for you. If you have very little antibody you can put your blot in a fresh autoclavable bag and seal it; thus you will use very little antibody.

Hi Himangshi,

There is no straight forward answer available to your question. Primary antibody dilution has to be standardized individually depending upon the species cross reactivity (immuno affinity) and actual stock antibody concentration. Monoclonal antibodies work better at high dilution compared to the polyclonal as well. Odyssey Li-Cor has a device that is widely used to determine antibody titer per channel (well in WB) using varied concentration of antibodies of interest.

The easiest way to overcome your problem is to begin with a higher dilution (may be 1:3000 or 1ug/ml you have to determine) for a longer duration of incubation at 4 deg C and you can play with your secondary antibody (increased concentration/ increased incubation time) as well. I am not sure what detection method you are using: Chemiluminiscence or IR?

If the higher primary dilution does not work then increase the concentration in a smaller increment to the same diluted antibody without throwing it away. I usually reuse the primary antibody dilution if kept carefully at refrigerator (4 deg C) without any contamination at least for a month.

To minimize the working volume of diluted antibody you can roll your NCP or PVDF into a 15 or 50 ml Falcon tube and add 2.5-3.0ml antibody dilution in the blocking buffer and use a roller mixer for incubation.

I hope these ideas work for you.

You should check product information of your antibodies. The dilution ratio of antibodies is various and depend on each one you used.

Yes, consulting product information sheet is a good idea and you can have some info about antibody dilution from validated experiments using proteins of interest from defined species .If your species of interest is not validated (say for example most of the cases it could be bovine/porcine) against the said antibody then you have to standardize the dilution on your own. Most of the primary antibodies usually come with validated data against rat, mouse, human etc.

The other possible ways to save antibody is try loading more protein for electrophoresis.

Consult the literature to see if anyone has published data using the same antibody. I tried for a while without success using Ab at manufacturer recommended dilutions in the past only to find that most published data featured dilutions well outside of the recommended range. Also, titration is a good way to determine. Select a range of primary concentrations with 2 or 3 secondary concentrations and pick the permutation which gives the clearest specific signal. Use well defined positive and negative controls where possible.

my rule of thumb is always making dilution series for the first instance upon receiving the antibody, using a protein that definitely works as the positive control. Sometimes the CoA states how much you should dilute, say 1:1000. I start with 1:500, 1:1000, 1:2500, 1: 5000, 1: 10,000. It just a matter of making series of dilution; do an overnight incubation at 4 DEG. I normally choose the highest dilution where I still can detect the signals (to save antibody). For further practice, just then load more protein (as already suggested) and give a longer exposure time upon development of the signal.

I agree with the previous responses. Unfortunately every antibody is different and the same antibody will behave differently under different conditions. Often the product sheets will have a concentration that is too high and increases background. A titration experiment is the best way to figure it out and the optimization is almost entirely empirical. Be sure you are using the proper blocking buffer conditions as well (including choice of buffer and detergents).