Quick ELISA for Fc tagged proteins secreted from mammalian cells.

This is a REALLY quick ELISA that will allow you to screen stables for expression in 96 well plates.

This assay is also VERY sensitive. It can detect protein in as little as 100ul of 20 fold diluted T.C. media and most importantly, does not recognize bovine IgG.

1. Obtain as many 8-well strips of ProteinG coated ELISA plates (Pierce #15133) as you need for your samples (Try to include at least 1 control [media with no protein] per strip).

2. Add either 100 ul of T.C. media or media alone to each well.

3. Incubate at least 30 minutes.

4. Wash the excess from plate with a plate washer (5 times).

5. Add anti-human IgG Fab antibody labeled with horseradish peroxidase (Rockland Immunochemicals #809-1302 [1:2500] in TBS or T.C. media).

6. Incubate an additional 30 minutes.

7. Wash the plate several (10) times with a plate washer.

8. Add 100 ul of mixed TMB substrate (Pierce #34021).

9. Incubate 10-15 minutes.

10. Add 100ul 2M Sulfuric Acid.

Read absorbance at 450 nm.