Immunofluorescence analysis of p38 MAPK was performed using 70% confluent log phase HeLa cells treated with 100 ng/ml TNF-alpha for 20 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with p38 MAPK alpha (6A1) Mouse Monoclonal Antibody (LFMA0126) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the untreated cells. Panel f represents the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Synthetic glutaldehyde peptide, KLH coupled, corresponding to the C-terminal residues of human p38 MAP kinase protein.|
|Storage buffer||HEPES with 0.15M NaCl, 0.01% BSA, 50% glycerol|
|Contains||0.03% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||3-5 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
A suggested positive control for this product is 293T cells.
p38 MAPK cascade regulates a variety of cellular responses to stress, inflammation and other signals. p38 MAPK is relatively inactive in the nonphosphorylated form and becomes rapidly activated by dual phosphorylation of a Thr-Gly-Tyr motifs. There are four isoforms of p38 MAPK, which differ in their tissue expression and affinity for upstream activators and downstream effectors. When cells are exposed to tumor necrosis factor, interleukin-1, heat shock, or other activating stimuli, activation of MAPK kinase-3 occurs by phosphorylation. Activated MAPK kinase-3/6 phosphorylate each residue of Thr180 and Tyr182 in p38 MAPK. Phospho-p38 MAPK activates ATF-2, CHOP-1, MEF-2 and other transcription factors through phosphorylation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.