The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 42, 44 kDa (predicted molecular weight: 42, 44 kDa).
Use a concentration of 5 µg/ml.
Involved in both the initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors such as ELK1. Phosphorylates EIF4EBP1; required for initiation of translation. Phosphorylates microtubule-associated protein 2 (MAP2). Phosphorylates SPZ1 (By similarity). Phosphorylates heat shock factor protein 4 (HSF4) and ARHGEF2. Acts as a transcriptional repressor. Binds to a [GC]AAA[GC] consensus sequence. Repress the expression of interferon gamma-induced genes. Seems to bind to the promoter of CCL5, DMP1, IFIH1, IFITM1, IRF7, IRF9, LAMP3, OAS1, OAS2, OAS3 and STAT1. Transcriptional activity is independent of kinase activity.
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily. Contains 1 protein kinase domain.
The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
Dually phosphorylated on Thr-185 and Tyr-187, which activates the enzyme. Dephosphorylated by PTPRJ at Tyr-187.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 42, 44 kDa Observed band size : 42,44 kDa
Exposure time : 4 minutes
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab94484 overnight at 4°C. Antibody binding was detected using a goat anti-rabbit HRP secondary antibody (ab6721), and visualised using ECL development solution ab133406
Western blot - ERK1 + ERK2 antibody (ab94484)
All lanes : Anti-ERK1 + ERK2 antibody - Loading Control (ab94484) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa nuclear extract lysate (TPA stimulated 2 hr) (ab14653) Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 6 : THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate Lane 7 : Human liver tissue lysate - total protein (ab29889) Lane 8 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 9 : Human kidney tissue lysate - total protein (ab30203)
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 42, 44 kDa Observed band size : 42,44 kDa Additional bands at : 120 kDa,130 kDa,35 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab94484 stained MCF7 cells. The cells were 4% PFA fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab94484, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa cells at 5µg/ml.