THE RECOMMENDATIONS BELOW REPRESENT GENERAL GUIDELINES AND ARE NOT MEANT TO BE ABSOLUTES. OPTIMIZATION OF ASSAY CONDITIONS IS THE RESPONSIBILITY OF THE RESEARCHER.
HBS: 8.76 grams of NaCl and 4.8 grams of Hepes in 900 ml of deionized water. Adjust pH to 7.4 with 10 N NaOH. Adjust the volume to 1 liter with deionized water. For antibody/antigen interactions that are calcium dependent, include 2 mM CaCl2 in the buffer. The frequent need for calcium containing buffers is the reason we do not use PBS for this and many other applications.
Antibody: Start with an antibody concentration of 100 nM and then adjust this concentration to optimize your specific application.
Incubations: Recommended incubation times are 30 to 60 minutes at room temperature or overnight (16 hours) at 4oC.Detection:For detection, we recommend using a FITC-labeled secondary antibody. These are readily available from a number of different suppliers, and we make no specific recommendations. Follow the manufacturer’s instructions for use of the FITC conjugate.