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Pre-clearing
Protein Lysate
Cell Lysis
Antibody
Protein A
Magnetic Beads
Binding
Washing
Protein of
Interest Elution
Cell lysis: The lysis step for IP should be relatively gentle to allow antibody-antigen binding, but also
harsh enough to efficiently extract proteins from the cells. Thus, it's important to choose a lysis reagent
that can guarantee high protein yields and retain protein activity.
Binding: Magnetic beads (or agarose), antibody, and antigen form complexes at this step. The buffers
used here and at the washing steps are vital to perform a successful IP. The order that these three
components are added also affect the results. Antibodies may be added first to covalently or
noncovalently bind to the magnetic beads before adding lysate, or incubated with lysate to form a
complex before adding protein A or protein G magnetic beads to purify the complex from the mixture.
Washing: Ideally, this step can break all nonspecific bindings without affecting the specific interactions
between antibody and antigen. Adding extra lysis buffer to the washing buffer can help breaking the
nonspecific bindings.
Elution: Generally, there are three kinds of elution buffers, including glycine, SDS, and urea elution. Any
of the three elution buffers can be used to elute the protein from the beads. The SDS buffer is harsh
enough to elute noncovalently bound antibodies, antibody fragments, and the protein of interest. The
glycine buffer is a gentler choice.
Troubleshooting Tips
1. To improve elution conditions
• Choose an appropriate lysis buffer according to the protein location (membrane, cytosolic, or nucleus).
The pH, detergent, salt, and divalent cation concentrations should be optimized for each IP.
• Check the antibody binding properties of each beads. It’s important to match the binding specificity of
the beads to the species and the antibody subtypes (Table 1).
Human IgM + - +
Human IgE ++ - ++
Human IgD - - -
Human IgA + - +
Human IgA1 + - +
Human IgA2 + - +
Human Fab + + +
Human ScFv + - +
Mouse IgG1 + ++ ++
Mouse IgM - - -
Rat IgG1 + ++ ++
Rat IgG2b - + +
Chicken IgY - - -
Hamster IgG ++ ++ ++
2. High background
• Remove supernatant immediately after centrifugations to avoid carryover of detergent-insoluble
proteins.
• To thoroughly wash the samples, place a lid on the tube and invert several times before
centrifugation.
• Pre-blocking beads with fresh BSA can decrease non-specific protein binding to the beads. Beads
are incubated with 1% BSA in PBS for an hour and then wash 3 to 4 times in PBS before use.
• Use an affinity-purified antibody with high specificity to avoid high background.
• Use recommended numbers of antibody or optimize the concentration of the antibody by titration.
Using too much antibody may cause non-specific binding.
• Decrease cell numbers to avoid high protein concentration in the lysate.
• In case of too much non-specific binding of proteins to antibody, use an irrelevant antibody of the
same species of origin and the same Ig subclass to pre-clear the lysate.
• Add fresh protease inhibitors in the sample lysate to prevent antigen degrading during IP.
• Inappropriate washing may cause high background. Use distilled water and increase the number of
washes. Before the last wash, it is a good practice to transfer the cell pellet to a new tube.