Protein Block

Protein Block has been developed to use with immunolabeling techniques for the reduction of nonspecific background staining while simultaneously reducing the handling of animal serums in the laboratory. The need to match species with the secondary antibody is eliminated due to the lack of normal serum in this product. Protein Block has been shown to be effective for immunohistochemical, ELISA, and blot and requires no mixing or diluting.

Protein blocking buffer ab64226 for serum-free blocking of non-specific antibody binding in IHC, ELISA and western blot. Ready to use. No mixing or dilution required.

For use in ELISA, add Protein Block to well and incubate for 2-10 minutes before addition of sample. Wash and continue procedure. For use in western blotting, incubate for one hour at room temperature or overnight at 4°C.

IHC protocol suitable for use with Protein Block ab64226:
For frozen sections, skip steps 1 and 2. For fluorescent IHC, skip step 3, incubate with fluorescent dye conjugated secondary at step 6, skip rest of steps, and mount with anti-fade mounting medium.

1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.

2. Use appropriate antigen retrieval buffer or enzyme (primary antibody dependent) to treat sections. Wash 3 times in buffer.

3. Add enough hydrogen peroxide blocking solution to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer. If necessary, use avidin biotin blocking.

4. Apply Protein Block ab64226 (or normal serum from same species as secondary antibody) and incubate for at least 30 minutes at room temperature to block nonspecific background staining. Wash once in buffer.

5. Apply primary antibody in antibody diluent and incubate. Wash 4 times in buffer.

6. Incubate with biotinylated secondary antibody (or HRP polymer secondary antibody and skip step 7). Wash 4 times in buffer.

7. Apply streptavidin-HRP and incubate for 10 minutes at room temperature.

8. Rinse 4 times in buffer. Place slide in DAB substrate or AEC substrate and incubate until desired color is achieved. Rinse 4 times in buffer.

9. Add enough drops of hematoxylin to cover the section. Incubate for 1 minute.

10. Rinse 7-8 times in tap water. Add mounting medium to cover the section.

Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.