[Histonet] Antibody dilution help

[richard cartun]

 Hi, Charles:
You may already have your answer by now, but I wanted to comment on preparing antibody dilutions.
Whenever I taught someone (or a group) how to prepare antibody dilutions, I emphasized two things: 1.) simplicity and 2.) consistency.  I have found over the years that many people struggle with dilution calculations because mathematics is involved, and, believe me, we all experience "Brain Fog" at one time or another when doing this.  I have always used a simple equation to calculate dilutions:
Dilution Factor = Total Volume (Diluent + Antibody) / Volume of Antibody            *** Make sure to use "mL" units for your calculations.

To solve this equation you must know two of the three variables ...... correct?  In your situation, the Dilution Factor is "100", and the Volume of the Antibody is "100 microliters" or 0.1 mL  If you multiply the Dilution Factor (100) by the Volume of the Antibody (0.1 mL) you get "10 mL" for the Volume of the Diluent.
Paula brought up a good point ...... some people will subtract the Volume of Antibody from the Total Volume (e.g., 99 parts of diluent and 1 part of antibody).  I never do this (opening the door for error) unless the volume of the antibody is equal to or greater than 100 microliters (0.1 mL).  Therefore, in your situation, I would add the 0.1 mL of antibody to 9.9 mL of diluent to yield 10 mL of a 1:100 dilution.
Let me ask you a question ....... Do you need 10 mL of diluted antibody?  If so, great, but let's say you only required 1.0 mL of diluted antibody, how would you solve the equation?
Dilution Factor (100) = Total Volume (1.0 mL) / "X" mL of Antibody
If you solve for "X" you would get 0.01 mL or 10 microliters.  Does that make sense?
I only used simple dilutions in my laboratory.  For example, if I need at least 1 mL of antibody:
1:5   (0.8 mL diluent + 0.2 mL antibody)
1:10   (0.9 mL diluent + 0.1 mL antibody)
1:20   (1.0 mL diluent + 0.05 mL antibody
1:50   (1.0 mL diluent + 0.02 mL antibody)
1:100   (1.0 mL diluent + 0.01 mL antibody)   
1:200   (1.0 mL diluent + 0.005 mL antibody)
1:500   (2.5 mL diluent + 0.005 mL antibody)
1:1,000   (5.0 mL diluent + 0.005 mL antibody) 

...... and we never encountered any problems.  If you have multiple people making dilutions in your laboratory, everyone has to do it the same way ...... be consistent!  Remember, the number of slides that you have to test should determine how much antibody you need and the pipettes available to you will determine what volumes you can pipette.

I hope this was helpful.
Richard W. Cartun, PhD, MSMorphologic Proteomics, LLC(860) 490-7633 Cell 
   On Friday, August 30, 2024 at 10:57:51 AM EDT, Charles Riley via Histonet <histonet at lists.utsouthwestern.edu> wrote:  
 
 Hello everyone,

Not sure if it's just burnout or Covid brain but I am having issues with
remembering the right way to dilute my antibodies, and all the calculators
online seem to give different responses.

I have 100 microliters of 1mg/mL antibody and I want to make a 1:100
dilution.

How much diluent do I add to do this?  And if anyone has any calculators
that they like using that would be awesome too
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