Our scientists are at the bench daily to produce and validate our antibodies, so they have hands-on experience and knowledge of each antibody’s performance.
The MAPK/Erk signaling cascade is activated by a wide variety of receptors involved in growth and differentiation including receptor tyrosine kinases (RTKs), integrins, and ion channels. GPCRs (G-Protein Coupled Receptors) activate the MAPK cascade using a different set of adaptors.
The specific components of the cascade vary greatly among different stimuli, but the architecture of the pathway is similar:
- Adaptors (Shc, GRB2, Crk, etc.) link the receptor to a guanine nucleotide exchange factor (GEF), i.e. SOS and C3G.
- The GEF proteins transduce the signal to small GTP binding proteins (Ras, Rap1).
- The GTP binding protein activates the core unit of the cascade composed of a MAPKKK (Raf), a MAPKK (MEK1/2), and MAPK (Erk). Activated Erk can regulate targets in the cytosol and also translocate to the nucleus where it phosphorylates a variety of transcription factors regulating gene expression.
Activation of Erk (extracellular signal regulated kinase) requires the phosphorylation of Thr202 and Tyr204 amino acid residues.
NIH/3T3 cell pellets (Figure A) treated with either TPA (to induce Erk phosphorylation) or U0126 (to inhibit Erk phosphorylation) (Figure B) paraffin embedded ovarian cancer sections.
- CST #4370 antibody shows strong cell staining in the TPA treated cells but not in the U0126 treated cells as expected.
- At a 1:400 dilution, Company 1 antibody shows weak staining in TPA induced cells. At a lower dilution/higher concentration, Company 1 antibody shows strong non-specific staining in the U0126 treated cells.
- CST #4370 stains ovarian cancer tissues more strongly than the company 1 antibody at the same dilution. When used at a lower dilution/higher concentration, company 1 antibody showed weaker yet comparable staining to CST #4370.
- Given the background staining by Company 1 antibody in the NIH/3T3 cell pellets treated with U0126, the ovarian cancer tissue staining specificity is questionable.
Western blot analysis of purified MAPK phospho-proteins or extracts from NIH/3T3 cells treated with UV light and PDGF, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) Rabbit mAb #4377 (upper), Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb #9215 (middle), and Phospho-SAPK/JNK (Thr183/Tyr185) (98F2) Rabbit mAb #4671 (lower).
Function: Activation of Erk (extracellular signal regulated kinase) requires the phosphorylation of Thr202 and Tyr204 amino acid residues.
Samples: Jurkat cells were treated with TPA, a phorbol ester that activates PKC to induce Erk phosphorylation.
- CST Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 recognizes the expected double bands at 44 and 42 kDa that correspond to Erk1 and Erk2 respectively.
- Company 1 phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) mouse monoclonal antibody recognizes the expected double band at 44 and 42 kDa but also shows high background and non-specific bands.
- Company 2 phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal antibody weakly detects the expected double band at 44 and 42 kDa. TPA strongly induces Erk phosphorylation, therefore the weak signal suggests low antibody binding.