HMGB1 antibody (N-Term)

Catalog No. ABIN2787911

Product Details anti-HMGB1 Antibody

Target: HMGB1 (High Mobility Group Box 1 (HMGB1))

Binding Specificity: N-Term

Reactivity: Human, Mouse, Rat, Cow, Dog, Rabbit, Guinea Pig, Horse, Zebrafish (Danio rerio)

Host: Rabbit

Clonality: Polyclonal

Conjugate: This HMGB1 antibody is un-conjugated

Application: Western Blotting (WB)

Sequence: QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKF EDMAKADKAR

Predicted Reactivity: Cow: 100%, Dog: 100%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%, Zebrafish: 93%

Characteristics: This is a rabbit polyclonal antibody against HMGB1. It was validated on Western Blot.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the N terminal region of human HMGB1

Application Details

Application Notes: Optimal working dilutions should be determined experimentally by the investigator.

Comment:

Antigen size: 215 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103662 (Western Blotting)

by: Lewis Lab, Department of Biology, College of Arts and Sciences, Syracuse University

No.: #103662

Date: 12/05/2019

Antigen: HMGB1

Lot Number: QC29977-40568

Method validated: Western Blotting

Positive Control:

Zebrafish embryos injected with Myc-Hmgb1a

Negative Control:

Uninjected embryos

Notes:

Passed. ABIN2787911 specifically recognizes Myc-Hmgb1a in embryos overexpressing this construct.

Validation Images

Full Methods

Primary Antibody: ABIN2787911

Secondary Antibody: IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 925-32211, lot C80925-01)

Full Protocol:

• Harvest >100 zebrafish embryos from wild type parents.
• Inject >50 embryos with 1ng mRNA encoding a Myc-tagged Hmgb1a construct. Leave the remainder of embryos uninjected as negative controls.
• Grow embryos to 6h post-fertilization.
• Enzymatically dechorionate all surviving, fertile embryos with 1mg/ml Pronase solution.
• De-yolk embryos mechanically by pipetting up and down several times, spinning down cells, and washing away soluble yolk proteins.
• Lyse each group of embryos mechanically with micropestle in 1µl per embryo of lysis buffer (50mM Tris-HCl pH8.0, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 1x Roche EDTA-free protease inhibitor cocktail, 1mM PMSF).
• Spin down debris and transfer lysates to fresh tubes.
• Mix lysates 1:1 with 2x Laemmli SDS sample buffer and incubate 5min at 95°C.
• Load 20µl of resulting mix per well of 4-20% Mini-PROTEAN TGX precast protein gel (Bio-Rad, 456-1095, lot 64268988) in a Mini-PROTEAN Electrophoresis Cell and run for 40min at 200V.
• Activate 0.45µm PVDF membrane (Millipore, product IPVH00010, lot K3HA8233EK) with methanol. Transfer proteins to membrane with transfer buffer (25mM Tris-HCl pH8.3, 192mM glycine, 20% v/v methanol) for 1h at 100V.
• Rinse membrane with water, then incubate 2min in PBS.
• Block the membrane with Odyssey Blocking Buffer (LI-COR, P/N 927-40000) 1h at RT with shaking.
• Incubate with either of primary
• • rabbit anti-HMGB1 antibody (antibodies-online, ABIN2787911, lot QC29977-40568) diluted 1:1000 in Odyssey Blocking Buffer (LI-COR, product no, lot no) containing 0.2% Tween 20 ON at 4°C with shaking.
  • mouse anti-Myc antibody (ThermoFisher Scientific, MA1-980, lot NJ17123) diluted 1:1000 in Odyssey Blocking Buffer (LI-COR, product no, lot no) containing 0.2% Tween 20 ON at 4°C with shaking.
• Wash membrane 5x 5min each with PBST (PBS, 0.1% Tween 20).
• Incubate with secondary
• • IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 925-32211, lot C80925-01) diluted 1:15000 in Odyssey Blocking Buffer containing 0.2% Tween 20 and 0.01% w/v SDS 1h at RT with shaking.
  • IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 925-32210, lot C70712-11) diluted 1:15000 in Odyssey Blocking Buffer containing 0.2% Tween 20 and 0.01% w/v SDS 1h at RT with shaking.
• Wash membrane 5x 5min each with PBST.
• Rinse membrane with PBS then hang to dry.
• Once dry, scan the membrane with LI-COR Odyssey CLX Scanner.

Experimental Notes:

• The Hmgb1 antibody ABIN2787911 reveals a protein of the expected molecular weight of the overexpressed Myc-Hmgb1a construct in the injected lysate. A Myc-tag antibody also detects this protein at the same size in the same lysate. This band is not detected by ABIN2787911 in an uninjected control lysate.

• ABIN2787911 also detects a faint band (or set of overlapping bands) at the expected size of endogenous Hmgb1a and Hmgb1b in both injected and uninjected lysates, but this band is weaker than several other non-specific bands.

• Protein amounts of Hmgb1a and Hmgb1a at this stage are unknown, so detection of these endogenous proteins was not necessarily guaranteed. Both corresponding mRNAs are expressed at this stage, so we predicted we might be able to detect the encoded proteins.

• Expected sizes of target antigens: are 25.1 kDa for pCS2-Myc-Hmgb1a, 23.7kDa for endogenous Hmgb1a: 23.7kDa, and 22.9kDa for endogenous Hmgb1b: 22.9kDa.

Handling

Format: Liquid

Concentration: Lot specific

Buffer: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.

Target Details for HMGB1

Target: HMGB1 (High Mobility Group Box 1 (HMGB1))

Alternative Name: HMGB1 (HMGB1 Products)

Synonyms: HMG1 antibody, HMG3 antibody, SBP-1 antibody, DEF antibody, HMG-1 antibody, Hmg1 antibody, amphoterin antibody, p30 antibody, hmgb1 antibody, ik:tdsubc_1a5 antibody, wu:fb23c02 antibody, xx:tdsubc_1a5 antibody, zgc:56110 antibody, zgc:77104 antibody, hmg-1 antibody, hmg3 antibody, sbp-1 antibody, hmg1 antibody, HMGB1 antibody, Ac2-008 antibody, high mobility group box 1 antibody, high-mobility group box 1 antibody, high mobility group box 1a antibody, high mobility group box 1 L homeolog antibody, high mobility group protein B1 antibody, HMGB1 antibody, Hmgb1 antibody, hmgb1 antibody, hmgb1a antibody, hmgb1.L antibody, LOC100359149 antibody

Background: Extracellular HMGB1 is an activator of human tumor cell migration operating in concert with EGF. HMGB1 encodes a protein that is potentially involved in the regulation of lipogenic and cholesterogenic gene transcription.
Alias Symbols: DKFZp686A04236, HMG1, HMG3, SBP-1
Protein Interaction Partner: AGTRAP, HUWE1, UBC, ASB2, ASB1, ZNF24, MAPK1, HMGA1, ITGA4, FN1, HNF1A, SSRP1, SMARCE1, PSEN1, XPC, XPA, RAD23B, CDK2, TFAP4, TERF2IP, TERF2, HHV8GK18_gp81, SUMO2, FCP1, DNM2, HSPA8, HMGB2, PDIA3, Ccdc15, POU5F1, H3F3A, CREBBP, CDK1, TBP, LRIF1, RASSF4, N
Protein Size: 215

Molecular Weight: 25 kDa

Gene ID: 3146

NCBI Accession: NM_002128, NP_002119

UniProt: P09429

Pathways: p53 Signaling, Regulation of Muscle Cell Differentiation, Skeletal Muscle Fiber Development, Positive Regulation of Endopeptidase Activity, Regulation of Carbohydrate Metabolic Process, Toll-Like Receptors Cascades, Smooth Muscle Cell Migration, Inflammasome