Question

what form/s of MAPK does the anti-total map kinase antibody detect, and what form/s of MAPK does the anti-active map kinase antibody detect?

Answer 1

Researches suggest that:

Mitogen-activated protein kinase (MAPK) are also known as the extracellular signal-regulated kinase (ERKs), they exist in two isoforms namely, ERK1 (MAPK3) and ERK2 (MAPK1).[12] ERK2 is mapped to human chromosome 22q11.2.[13] ERK1 is located on human chromosome 16p11.2.[14]

Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody, Mouse monoclonal (mouse IgG1 isotype) is derived from the MAPK-YT hybridoma produced by the fusion of mouse myeloma cells (NS1) and splenocytes from BALB/c mice immunized with a synthetic phosphorylated peptide containing amino acids corresponding to the phosphorylated form of ERK-activation loop, conjugated to KLH. The isotype is determined by a double diffusion immunoassay using Mouse Monoclonal Antibody Isotyping Reagents, Product Number ISO2.

Specificity

Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) is specific for the active, dually-phosphorylated form of MAP kinase (ERK-1 and ERK-2, 44 kDa and 42 kDa, respectively). Reactivity has been observed with human, bovine, rat, mouse, hamster, Xenopus, Drosophila, Spodoptera frugiperda, C. elegans, and yeast.

The antibody reacts specifically with the diphosphorylated form of MAP kinase (ERK-1 and ERK-2). It does not recognize the non-phosphorylated or the monophosphorylated forms of MAP kinase or the diphosphorylated forms of JNK and p38 MAP kinase. The epitope recognized by the antibody contains the phosphorylated threonine and tyrosine residues within the regulatory site of active MAP kinase.

Immunogen

synthetic peptide HTGFLpTEpYVAT corresponding to the phosphorylated form of the ERK-activation loop.

Application

The antibody may be used for immunoblotting applications at a working concentration of 0.5-1.0 μg/mL in cultured cells as well as tissue extracts. A working dilution of 1:80-1:100 is suitable for immunohistochemistry (formalin and formaldehyde-fixed sections). The antibody may also be used for immunocytochemistry, immunoprecipitation, indirect ELISA and protein microarray.

Monoclonal Anti-MAP Kinase, activated (Diphosphorylated ERK-1&2) antibody produced in mouse has been used in detection of

• ERK1/2 in cardiac myocytes using immunofluorescence microscopy

• ERK1/2 in human eosinophils using western blotting

• ERK in human bronchial epithelial cells using protein array

• ERK proteins in human liver cell lines using immunofluorescence assay and western blot analysis

• ERK1/2 in human embryonic kidney cells by immunoprecipitation

Physical form

Solution in phosphate buffered saline, pH 7.4, containing 15 mM sodium azide.

Storage and Stability

For continuous use, store at 2-8 °C for up to one month. For extended storage, freeze in working aliquots at -20 °C. Repeated freezing and thawing, or storage in frost free freezers, is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use. Working dilutions should be discarded if not used within 12 hours.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Biochem/physiol Actions

Mitogen-activated protein kinase (MAPK) superfamily of enzymes is involved in widespread signalling pathways.[7][3][4] Members of this family include the ERK1/2 (extracellular signal-regulated protein kinase, also termed p42/p44 MAPK), JNK and p38 MAPK subfamilies. These are the terminal enzymes in a signalling cascade where each kinase phosphorylates and activates the next member in the sequence. Phosphorylation of both tyrosine and threonine is essential for the full activation of all MAPKs.[2][11][9][8] Several kinases participate in activation of the ERK cascade. This cascade is initiated by the small G protein Ras, which upon stimulation causes activation Raf1 kinase. Raf1 continues the transmission by activating MEK. Activated MEK appears to be the only kinase capable of specifically phosphorylating and activating ERK.[1] ERK appears to be an important regulatory molecule, which by can phosphorylate regulatory targets in the cytosol (phospholipase A2, PLA2), translocated into and phosphorylate substrates in the nucleus (ELK1). The activation of ERK cascade mediates and regulates the signal transduction pathways in response to stress, mitogenic signals and is important in development and differentiation, learning, memory and survival.[6][5]

Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK-1 and 2) is specific for the active, dually-phosphorylated form of MAP kinase (ERK-1 and ERK-2, 44 kDa and 42 kDa, respectively). The epitope recognized by the antibody contains the phosphorylated threonine and tyrosine residues within the regulatory site of active MAP kinase (Thr183 and Tyr185 in ERK-2). It does not recognize the non-phosphorylated or the monophosphorylated forms of the MAP kinase molecule or the diphosphorylated form of Jun-kinase (JNK) and p38 MAP kinase.

Signaling pathways mediated by MAPK are associated in the pathogenesis of neurodegenerative disorders and cancer.[15] Deletion in the ERK1 gene locus is associated with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS)[13] and congenital heart defects. Rearrangements in MAPK3 gene locus may contribute in the pathogenesis of autism spectrum disorders and schizophrenia.

Other research suggest that:

Anti-ACTIVE® MAPK pAb is a polyclonal rabbit antibody that preferentially detects the dually phosphorylated, active form of the mitogen-activated protein kinase (MAPK) enzymes (ERK1 and ERK2). The antibody is affinity purified using a dually phosphorylated peptide that corresponds to the active form of the mitogen-activated protein (MAP) kinase enzymes.

When used at the recommended 1:5,000 dilution, this product will generate 200ml of blotting solution, sufficient for 20 Western blots of 10ml each.

• Immunogen: Dually phosphorylated Thr/Glu/Tyr region (pTEpY) derived from the catalytic core of the active form of the mitogen-activated protein kinase (MAPK) enzymes, ERK1 and ERK2, which corresponds to Thr183 and Tyr185 of the mammalian ERK2 enzyme.
• Antibody Form: Affinity-purified rabbit IgG; supplied in PBS (pH 7.4).
• Working Dilutions: Western blotting (1:5,000); Immunocytochemistry (1:500); Immunohistochemistry (1:500).

Here are the fulltext document:

https://worldwide.promega.com/-/media/files/resources/protcards/anti-active-mapk-p38-and-jnk-polyclonal-antibodies-quick-protocol.pdf?la=en

https://worldwide.promega.com/-/media/files/resources/protocols/technical-bulletins/0/anti-active-mapk-jnk-and-p38-polyclonal-antibodies-protocol.pdf?la=en

Explanation:

Here are some of the references of the answers I have provided. You can also check their articles to add more ideas in your assignment.

Thank you and Godbless!

Always be happy to learn :)

https://www.sigmaaldrich.com/catalog/product/sigma/m9692?lang=en&region=US

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017500/

https://www.biocompare.com/pfu/110447/soids/429320/Antibodies/MAPK

https://www.cellsignal.com/products/primary-antibodies/p44-42-mapk-erk1-2-antibody/9102