MEK3/MEK6 Rabbit Recombinant mAb

Catalog No.A5050

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  1. 1. Why choose rabbit monoclonal antibody?
  2. Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
  3. 2. Why choose recombinant antibodies?
  4. Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.

WB

Validated by Selleck

IF

Validated by Selleck

Usage Information

Application WB, IF,ELISA
Dilution
WB IF
1:1000 1:100
Reactivity Human Mouse Rat
MW (kDa) 39,37kDa
Source Rabbit
Concentration 1mg/ml
Storage buffer 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
Storage Store at –20°C.

Protocol

WB
+ Expand

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

IF
+ Expand

Immunofluorescence (Immunocytochemistry)

Specimen Preparation (forcultured cell lines, IF-IC)

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
2. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Mount slides usingmounting medium with DAPI and cover with coverslips.
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Description

Specificity

MEK3/MEK6 Antibody detects endogenous levels of MEK3/MEK6.

Background

Four distinct MAP kinase signaling pathways involving 7 MEK enzymes have been identified. MEK3 and MEK6 are functionally similar and encoded by MAP2K3 and MAP2K6 genes, respectively. The genes are both located on chromosome 17q. MEK3 and MEK6 consist of 347 and 334 amino acids residues respectively. Structurally MEK6 differs from MEK3 in terms of C- and N- terminal regions. However, the ATP binding sites, and serine/threonine and tyrosine catalytic sites are conserved. MEK3/6 signaling pathway is activated by growth factor stimulation through RTKs. Additionally, the cascade can also be activated by G-protein coupled receptors, intracellular receptors, and toll-like receptors, in response to numerous stimuli including physical and chemical stresses, hormones, UV irradiation, and cytokines, such as interleukin-1 and tumor necrosis factor. These stimuli activate different MAPK kinase kinases (MAPKKKs), which include TAK1, ASK1/2, DLK, MEKK4, TAO1/2/3 and MLK2/3. Active MAPKKKs phosphorylate and activate MEK3/6, which in turn catalyzes the concomitant phosphorylation of a threonine/serine and a tyrosine residue in the p38 MAPK. MEK6 activates all the four isoforms of p38 MAP kinase (α, β, γ and δ) whereas MEK3 can only activate p38α and p38β isoforms. MEK3/6-p38 MAPK cascade promotes p53-dependent growth arrest by phosphorylating p53 at serine 33 and 46. Together, these targets of MEK3/6-p38 MAPK pathway (cyclin D1, Cdc25, and p53) cooperate to arrest the cell cycle.

References

Datasheet & MSDS

Related Antibodies

MEK Signaling Pathway Map

Related MEK Products

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