Good day! I have a question. This is my first time that I am working with a primary antibodies. What can I use as a diluent for this? PBS?
In most simplified way, just estimate first that how much total volume of your primary antibodies you may need for your assay from your available stock. For example; lets say you need a total 3 ml of your primary antibodies, which also means 3000 uL. Now you need them to be diluted as 1:1000, so simply divide 3000 / 1000 which is 3. So take 3 uL from your Primary antibodies stock vial and add into 3000 uL (3 mL) of PBS or any other diluent as per your choice. So this is yours 1:1000 dilution in total of 3 ml. To confirm this calculation, just divide 3000 / 3 which gives 1000 which is our desired dilution factor here. You can use this simple way to dilute whatever and whenever you like, but you must sure about the total volume you need to work with in order to keep safe your stock and not to waste it by any wrong calculations.
The preferred buffer for diluting antibodies will depend on the application. Immunohistochemical labeling procedures frequently use PBS or HBSS or some similar phosphate-based buffer, usually at pH 7.2-7.4. These buffers are often supplemented with normal serum from the species that the secondary antibody was raised in, BSA and a detergent. Our standard buffer for diluting antibodies for immunolabeling is:
2-10% normal goat serum + 5% BSA + 0.5% triton X-100 in HBSS, pH7.4 (PBS will also work just fine).
A number of western blot protocols call for Tris buffers.
Hello, it depends mainly on the protein you are going to detect...if it`s a small size protein you should use a concentration equal or below the 1:1000...best thing to do in this case is to check your antibody information report (the one given by the company you bought from) there you will find the optimal dilution o use your antibody
In most simplified way, just estimate first that how much total volume of your primary antibodies you may need for your assay from your available stock. For example; lets say you need a total 3 ml of your primary antibodies, which also means 3000 uL. Now you need them to be diluted as 1:1000, so simply divide 3000 / 1000 which is 3. So take 3 uL from your Primary antibodies stock vial and add into 3000 uL (3 mL) of PBS or any other diluent as per your choice. So this is yours 1:1000 dilution in total of 3 ml. To confirm this calculation, just divide 3000 / 3 which gives 1000 which is our desired dilution factor here. You can use this simple way to dilute whatever and whenever you like, but you must sure about the total volume you need to work with in order to keep safe your stock and not to waste it by any wrong calculations.
you should add a little protein [ie. BSA or serum (from another species than what you are testing)] for blocking purposes in your experiment as well as to help stabilize the antibody. Additionally, you may want to add a little detergent (0.05 -0.1% Tween-20) to cut down on nonspecific antibody.
I agree with you Jawward except that I see you exceed the volume of 3 mL. It should be 2997 uL of the diluent and 3 uL of antibody
The diluent depends on the Antibody. Generally the anti body information sheet would provide you with that information. Once you know that you can dilute the stock concentration with the help of the simple formula.
Amount of stock required=(Strength Required/ Stock strength) X Volume required (diluent)
So if you are doing a 1:1000 dilution for 10ml of your diluent you need to add 10ul from the stock.
Sikozile Ncembu Good observation to be exact... But, most times we take Jawwad Ahmad option without being precise and it works fine. But it is better to follow your choice especially for the beginners in this area knowing well that some Abs are bizarre to deal with in many occasions.
The antibody you have purchased will usually come with recommendations for its use, but if it doesn’t, a good place to start is at 1 μg/mL, which is usually a 1:1,000 dilution from your tube if the primary antibody concentration is 1 mg/mL. That means for 1 mL of staining solution, you would add 1 μL of antibody to 1 mL of PBS or blocking solution.
Does this dilution hold true when the stock antibody comes in a concentration of 200ug/mL?
Cody,
That will depend on your specific antibody and antigen. I would recomend trying several different dilutions to see what works best for your purposes. Also, you might check the manufacturer's datasheet and literature references using the same antibody to get some idea of what dilution range has worked well for other investigators.
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