Exalpha Biologicals, Inc.

	Phospho-Erk1/2 ELISA Kit Phospho-Erk1 (T202/Y204) Phospho-Erk2 (T185/Y187)
The Exalpha Biologicals, Inc. Phospho Extracellular signal-Regulated Kinase 1/2 (ERK 1/2) ELISA is a non-isotopic immunoassay for the in vitro quantitation of human phospho ERK 1/2 (T202/Y204-T185/Y187) protein in cell lysates. Also called p42/p44 or Mitogen Activated Protein Kinase 1/2 (MAPK1/MAPK2).

Features - Colorimetric assay - HTS compatible format - High sensitivity - 4 deg C storage with long shelf life - Non-radioactive - 5 hour protocol	Performance Summary - Sensitivity: 40 cells/well - Intra-assay C.V.'s: <3% Mid-level - Inter-assay C.V.'s: <4% Mid-level
	Ordering Information
	Cat. No. X1839K (pdf)	Size 1 Plate
	For larger quantity pricing, please contact Customer Service

Legend: CHO cells transfected with the human S1P4 receptor for S1P (Edg6) were serum stared for 4 hours and challenged with S1P or LPA. PMA was used as a positive control for Phospho ERK 1/2 production. PHOSPHO ERK 1/2 was detemined to be an excellent surrogate marker for stimulation with S1P compared with traditional methods for GPCR signaling such as intracellular Calcium flux.

Protocol Summary: Not to be used in place of detailed protocol. See kit manual for complete protocol.

Steps	Incubation
1. Equilibrate kit to room temperature	-
2. Add samples and standards to wells	2 hours room temp. (or overnight 4°C)
3. Wash 4x	-
4. Add detector antibody to all wells	2 hours - room temp.
5. Wash 4x	-
6. Add HRP conjugate to all wells	30 minutes - room temp.
7. Wash 4x	-
8. Add substrate to all wells	30 minutes - room temp.
9. Add stop solution to all wells	-
10. Read plate at 450 nm/550 nm	-