Is the monoclonal antibody a mouse IgG1? the yield would be low and it would be important to check the final purity of your preparation, for example by running and SDS-PAGE, to check that you are not getting a lor of other proteins from the hybridoma culture medium

Its IgG2..However will try run it on SDS-PAGE. Thank you

You could try ammonium sulphate precipitation instead of protein A, if need be followed by HIC or SEC. 

My Guess from the information provided is that you are recovering little S9.6 antibody for the reasons below.

Your value for the extinction coefficient looks very high (10 X high) - Most of the monoclonal Ab I have worked with are ~1.4 A280/1% solution.  So when you calculate your dilutions you are going to be about a log low and miss your activity reading.   You need to run a gel for purity as mentioned above.  You may not be getting good washing on your spin column may want to increase to 5 washes of 1 column volume each then elute using a 1 column volume of elution buffer.  These spin columns are generally 0.5 - 0.75 mL each. 

When using Protein A, I have had at least 250 mM NaCl (High Salt up to 750 mM) in my binding buffer - the urban folktale is that high salt maximizes the Fc conformation that promotes more efficient binding to the Protein A receptor.  The Thermo Scientific (Pierce) Protein A binding buffer works really well and save you time in buffer formulations.

What are the yields of your Ab in the tissue culture supernatant - Common Range 10 - 100 ug/mL of culture supernatant?

Frankly, to increase your chances of success I would sequester 10 ml (minimum) of the tissue culture supernatant and run that over a 1 mL Protein A Hi-Trap column.  Your Ab yields will range between 100 -1000 ug.   Ab concentration ranges between 0.1 - 1.0 mg/mL. 

Good Luck - looking forward hearing of your successes.

Hi All, 

Thank You for the suggestion and will work on it and update if i have successfully purified it. 

* My sample is from ascites fluid which was collected about 5-10ml but we are planning to give a try on purifying the antibody obtain from  tissue culture supernatant as well. 

Thank You. 

Hello!

you said that you can performe immunofluorescence of the S9.6 antibody in vivo and obscure it under microscope. I am just wondering that if the Fab fragment of S9.6 antibody can be labeled with GFP and how you transform it to nucleus.

waiting for your anwsers, thanks!