This kit contains immobilized Protein G Beads, Binding/Wash Buffer, Elution Buffer (gentle elution buffer of high ionic strength is available separately), Handee Spin Cup Columns and microfuge tubes, lane marker sample buffer and Mammalian Protein Extraction Reagent. I have used this kit for immunoprecipitation of a 63 kDa protein from mammalian cell extracts. I plan to use it further for pull-down assays. I have already consumed all the columns and have ordered more Protein G beads, which are available in slurry form in order to prepare new columns and do further immunoprecipitations. Since a vial of cross-linker can’t be used again, if we are using less Protein G for a column, we ultimately fall short of the cross-linker vials. So smaller aliquots of the cross linker is suggested to the manufacturer.
I did immunoprecipitation for the first time with this kit and I got results in my first shot, so I will highly recommend it for beginners. The cross linking of the antibody is achieved by allowing antibody to bind in the presence of a cross linker (DSS) which is highly hygroscopic. The cross-linking of the antibody is crucial and very useful for probing the desired protein, especially when it lies in the range of the molecular weight of the heavy chain of IgG antibodies (~50 kDa). Since there is no antibody contamination, the desired band generates a clear signal.
Since all the components are present in 1 kit and the protocol is easy to follow, with 1 minute room temperature centrifugation steps, this kits makes a good choice for a fast and simple IP experiment. I have not determined the binding efficacy of the antibody to the Protein G beads but there is a considerable amount of antibody present in the washes.
High elution volumes require the concentration of the eluted sample which is tedious and not desired if downstream applications of functional activity assays are to be done. If using gentle elution buffer for elution, the high salt concentration poses a problem in concentrating. That adds a step of dialysis which may further dilute the eluted sample.
The columns cannot be reused as many times as claimed. I have found that the regenerated slurry, when incubated at 4ºC for 1 month, forms some kind of precipitates which makes the wash and elution steps tedious. You need to use high speed and longer spins to get these steps done.