HMGN (phospho-S20/S24) antibody

• Catalog Number: orb345524
• Category: Antibodies
• Description: HMGN (phospho-S20/S24) antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, IHC, WB
• Reactivity: Human
• Isotype: IgG
• Immunogen: This affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to amino acids 19-28 of human HMGN protein (see below).
• Concentration: 1.20 mg/mL
• Dilution range: ELISA: 1:5,000 - 1:25,000, IHC: 20 µg/ml, WB: 1:500 - 1:2,000
• Form/Appearance: Liquid (sterile filtered)
• Purity: This affinity-purified antibody is directed against phosphorylated human HMGN protein at pS20 and pS24 residues (other HMGN proteins have this conserved sequence but at other residue positions). The product was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross-adsorbed against the non-phosphorylated form of the immunizing peptide. Reactivity occurs against human HMGN pS20/pS24 proteins and the antibody is specific for the phosphorylated form of the proteins. Reactivity is seen against HMGN1 and HMGN2. HMGN3 is also likely reactive although not tested. Reactivity with non-phosphorylated human HMGNs is minimal by ELISA. A BLAST analysis was used to suggest cross reactivity with HMGN proteins from Xenopus, chicken, mouse, bovine, dog, orangutan, rat and swine based on 100% homology with the immunizing sequence. Reactivity against homologues from other sources is not known.
• Conjugation: Unconjugated
• UniProt ID: P05114
• NCBI: 386779
• Storage: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: rabbit anti-HMGN pS20/pS24 antibody, Non-histone c
• Note: For research use only
• Application notes: Anti-HMGN pS20-pS24 affinity purified antibody has been tested for use in ELISA, immunohistochemistry, and western blot. Specific conditions for reactivity should be optimized by the end user. Expect a band approximately 17 kDa in size corresponding to phosphorylated HMGN proteins by western blotting in the appropriate cell lysate or extract. Less than 0.5% reactivity is observed against the non-phosphorylated form of the immunizing peptide. This antibody is phospho specific for pS20 and pS24 of HMGN proteins. The antibody was tested against HMGN1 by performing a standard phosphorylation in vitro assay using HMGN1 as a substrate and Msk1 as a kinase (known as a main effector kinase of MAPK signaling pathway and in vivo kinase for HMGN1). This kinase specifically phosphorylates S20 and S24 of HMGN1 (S24 and S28 of HMGN2). The reaction was resolved on SDS PAGE and immunoblot, using antibody diluted at 1:1,000 and 1:5,000. The antibody shows strong positive signal for Msk-phosphorylated HMGN1 and no cross-reaction for non-treated HMGN1.
• Expiration Date: 12 months from date of receipt.

Biorbyt's affinity purified anti-HMGN pS20/pS24 antibody was used at 20 ug/ml to detect signal in a variety of tissues including multi-human, multi-brain and multi-cancer slides. This image shows moderate nuclear and faint cytoplasmic positive staining of epidermal keratinocytes at 40X. Tissue was formalin-fixed and paraffin embedded. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain.

Western blot using Biorbyt's Affinity Purified anti-HMNG antibody shows detection of phosphorylated HMGN1 and HMGN2. Recombinant native and mutant HMGN proteins were treated with kinase PKCalpha to specifically phosphorylate S20 and S24 residues. Lanes contain: 1 - HMGN1, non-phosphorylated, 2 - HMGN1, phosphorylated, 3 - HMGN1 delta20E, delta24E, non-phosphorylated, 4 - HMGN1, delta20E, delta24E, phosphorylated, 5 - HMGN2, non-phosphorylated, and 6 - HMGN2, phosphorylated. Molecular weight markers (not shown) confirm the size of each recombinant protein. The primary antibody was diluted 1:1000 for this experiment. The blot was processed using a 5 sec exposure time.