How to Block Buffers

Block buffers are used to stabilize coated proteins in immunochemistry assays and, according to Immunochemistry Technologies, they reduce noise in the results of individual assays, increase the sensitivity of the assay, increase the efficiency and shelf life of the coated plates and enhance reproducibility. Block buffers inhibit nonspecific binding after the proteins and antibodies have bound together and been absorbed onto the coated assay plate during an ELISA (an enzyme-linked immunosorbent assay). Because ELISAs can be built in different ways, there are several choices of blocking buffers to meet a technician's individual requirements.

Things You'll Need

• Antibody sample
• Protein sample
• Assay plate
• Blocking buffer
• Laboratory beakers
• Pipette

Show More

Instructions

• 1

  Select your blocking buffer depending on the type of ELISA you will be performing. Choices of buffer include: general blockers (mammalian protein scaffold) for sandwich and antigen-down ELISAs; BB2 neptune blocking (non-mammalian protein scaffold) for human and mammalian species ELISAs; BB3 synblocks (small, synthetic molecular scaffolds) for high-strength blocking ELISAs; and BB4 phosph-free (small, synthetic, phosphorus-free molecular scaffolds) for high, non-specific enzyme-labeled conjugate binding ELISAs. The BB4 buffer blocker is specifically useful in assays that use alkaline phosphatase, chemically-treated or glass reaction plates and in microfluidics, as reported by Immunochemistry Technologies, a company that supplies all relevant assay equipment.

• 2

  Coat the assay plate with your antigen (protein) or the antibody by pouring the solution across the plates.

• 3

  Aspirate (remove) the coating solution by tipping the plate so the excess is discarded into a separate container.

• 4

  Pipette 300 to 400 microliters of the blocking buffer into each well of the assay plate using a pipette.

• 5

  Incubate the solutions for around three hours or overnight at ambient temperatures (4 degrees Celsius). According to the Cell Technology company, the right block buffer will conserve your reagents, which can be valuable because they are costly and can be in short supply.

• 6

  Wash the excess protein to remove excess amounts as this may reduce the detection of your target antigen, as reported by Pierce Net.

• 7

  At this point, the reaction is complete and you should have a finished plate containing the right amount of antibody bound to antigen, which will give consistent results and reduce plate-to-plate variation in large, comparative assays.

• 8

  In the case of a microarray, use an advanced blocking solution such as Array It's Block It Buffer, which has been designed specifically to inactivate any reactive groups that remain following a "print" of substrates on slides containing super-epoxy, super-amine and super-aldehyde while retaining sharpness of resultant signal, preventing fluorescence and facilitating noise reduction. This reagent has a shelf life of one year if correctly stored, is extremely pure and is provided as a ready-to-use concentrate.