How to remove cross-reactivity to Rubisco of a primary antibody?

There are different reasons why a primary antibody may also recognize Rubisco, namely:

• The antigen that was used to elicit a given antibody shares epitopes with Rubisco
• The antigen preparation was contaminated by Rubisco
• Rabbits are routinely fed with plant based diet and may develop antibodies to Rubisco during the immunization program. To minimize this risk, it is advised to check pre-immune serum before the immunization is started. If the serum from non-immunized animals already shows strong reactivity to Rubisco, those animals should not be used in antibody production to a plant proteins. Exceptions are if the MW of the target protein is 30 kDa lower or higher than Rubisco (for Western blot application), or if the analyzed tissue does not contain Rubisco protein.

If a produced antibody shows cross-reactivity to Rubisco, so called purification by depletion protocol can be applied. The steps of the protocol to remove cross-reacting antibodies are the following: 

• Prepare a cell extract from young leaves. 
• Separate it on a gel (12 or more wells, or a gel with one wide well only).
• Use as high of a protein concentration/well as possible.
• Conduct protein transfer applying optimal conditions for transfer of 55 kDa protein.
• Cut out the part of a membrane with Rubisco transferred to it. 
• Incubate your antibody preparation (in TBS-T with a blocker) with the Rubisco membrane for 2 h/RT with agitation.
• Collect the primary antibody preparation and test its cross-reactivity to Rubisco. 

The Rubisco membrane can be stripped and re-used up to 10 times.