Haynes:ELISA

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ELISA Assay
Enzyme-linked immunosorbent assay
by Karmella Haynes, 2013

Principle: Proteins are captured on the bottom of a micro well plate, either by direct binding or by a conjugated antibody "trap". A second antibody is added to detect one specific type of protein. A counter-stain antibody (usually HRP-conjugated) is used to generate visible signal, which is proportional to the number of proteins. Normalization (e.g., using the number of cells per lysate sample, and a purified protein with known concentration if you're fortunate to have one available) can be used to calculate proteins per cell.

DIRECT ELISA

This key feature of this approach is the attachment of proteins directly to the bottom surfaces of the micro wells. A specific protein is detected with a primary and secondary-HRP. The advantage over "sandwich" ELISA is that fewer antibodies are needed, but this method is known to be less sensitive/ accurate. Because of this I recommend this method for artificially over-expressed or abundant naturally expressed proteins.

MATERIALS

• Well-Coated Amine Binding, 8 well strip plate, clear (G Bioscience #786-753)
• FemtoELISA-HRP kit (G Biosciences #786-110)

PROCEDURE

Cell lysis

1. Standard protein prep procedure

Bradford assay

1. See the Haynes:Bradford Bradford Assay Protocol

Protein-well binding

Primary antibody

Secondary antibody-HRP

What to do with your data: calculate unknown protein concentration(s) per cell