How do you dilute secondary antibodies?
A good starting concentration for a typical secondary antibody in that concentration range would be a dilution of 1:1,000. If you find your staining to be extremely bright, or that you have too much background, you can always try a higher dilution (from 1:2,000 to 1:10,000).
How do you dilute a secondary antibody for a western blot?
To correctly dilute secondary antibodies for western blotting it is recommended to first make an antibody working stock solution of 1 µg/mL and dilute further for the final antibody incubation solution.
What is the secondary antibody concentration for Western blot?
AP conjugated secondary antibodies can be routinely used at dilutions ranging from 1/5,000 to 1/50,000. Based upon a 1/5,000 dilution you can perform 1,500 blots with a single vial of an AP conjugated secondary. HRP secondary antibodies can be used at dilutions ranging from 1/2,000 to 1/20,000.
How do I make a 1 10000 dilution?
Another way is to dilute the stock 1/10 twice and then perform a further 1/100 dilution: 1/10 x 1/10 x 1/100 = 1/10,000 dilution This would yield 100 ml of a 1/10,000 dilution of stock in water.
How do you dilute antibodies?
The diluent is usually a phosphate buffer or buffered saline at a particular pH with or without detergent. This ratio can be applied to different volumes depending on how much is needed. For example, if we need 200 μL of a 1:50 solution, then we will add 4 μL of antibody to 196 μL of diluent.
How do you do a 1 1000 dilution?
You could make 1/1,000 by adding 1 microliter of sample to 0.999 ml diluent. Why is that a poor choice? Because you can’t measure 1 microliter (or even 10 microliters) accurately with ordinary pipeters. So, make three serial 1/10 dilutions (0.1 ml [100 microliters] into 0.9 ml): 1/10 x 1/10 x 1/10 = 1/1,000.
How long can you leave secondary antibody on?
How long should you incubate with secondary antibody in a Western Blot? Usually 1-2 hours at room temperature or overnight at 4°C , with agitation.
How do you dilute 100 times?
For a 1:100 dilution, one part of the solution is mixed with 99 parts new solvent. Mixing 100 µL of a stock solution with 900 µL of water makes a 1:10 dilution. The final volume of the diluted sample is 1000 µL (1 mL), and the concentration is 1/10 that of the original solution.
How much antibodies do I add?
Generally increase it 2-5 fold, but even 1x will often be enough. If you routinely stain 100 million cells (for a sort, for example), it might be useful to do a quick, 2- or 3-point titration on that many cells. Likewise, if you stain only 50,000 cells, do NOT DECRASE the antibody amount.