Determination of cross-reactivity of GFP-A1(Resin) for various fluorescent proteins


Overview
Abstract
This protocol details how to determine whether GFP-A1(Resin) cross-reacts with various fluorescent proteins. Briefly, equimolar solutions of purified proteins are incubated with equivalent aliquots of the Resin, the resin is washed, the bound protein eluted with low pH, and the eluants analyzed qualitatively by SDS-PAGE.
Type
  • Immunoprecipitation
Target
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Dynamic Range
Acceptable
Results
Summary
This application demonstrates that GFP-A1(Resin) can immunoprecipitate not only EGFP, but also Blue Fluorescent Protein (BFP). The resin did not immunoprecipitate Cyan Fluorescent Protein (CFP), DsRed, mCherry, Yellow Fluorescent Protein (YFP), or the negative control BSA. The proteins used in this application were purchased from BioVision (Cat# K816-6-100).
Figure

Image 

Gel for cross-reactivity of nanoCLAMP GFP-A1

Legend 

SDS-PAGE of immunoprecipitation of various fluorescent proteins with GFP-A1(Resin). Purified proteins were prepared at equimolar concentrations and incubated with 10 ul of GFP-A1(Resin) for 2 hours, washed, and eluted. Panel A: SDS-PAGE analysis of eluants of the GFP-A1(Resin) incubated with: lane 1: EGFP, lane 2: BFP, lane 3: CFP, lane 4: dsRed, lane 5: mCherry, lane 6: YFP, lane 7: BSA.  Panel B: 83 pmol of the protein solutions prior to incubating with the resin (approximately 2.5 ug per lane). Proteins are in the same order as in A.