Abstract
This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize certain 3D structure in solution of antigens might not be purified using such a method. Regarding this issue, the flow through is recommended to be kept and can be used for other purification methods with folded antigens.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Kurien (2009) and developed in Guowei Fang’s lab, Departmental of Biological Sciences, Stanford University, CA, USA. Funding from the NIH supported this work.
References
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"BCA neither shows higher concentration of protein in comparison to [elution buffer with Tris]. "Did you elute with Glycine or Tris? Tris will not elute the protein at all. If you used Glycine, have you checked the pH which is critical for the elution.