HMGB1/HMG-1 Antibody - BSA Free

Novus Biologicals | Catalog # NB100-2322

Novus Biologicals
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Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat, Bovine, Canine, Rabbit, Sheep

Cited:

Human, Mouse, Rat, Bovine, Rabbit

Predicted:

Equine (100%), Porcine (100%). Backed by our 100% Guarantee.

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, ELISA, Flow Cytometry, Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Simple Western, Knockdown Validated

Cited:

Western Blot, ELISA, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all HMGB1/HMG-1 Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to an internal portion of the human HMGB1 protein sequence (between residues 100-200). [UniProt #P09429]

Reactivity Notes

Rabbit reactivity reported in scientific literature (PMID: 27401639).

Localization

Nuclear

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

29 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for HMGB1/HMG-1 Antibody - BSA Free

Knockdown Validated: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Western blot shows lysates of HEK293T human embryonic kidney parental cell line and HMGB1 knockout (KO) HEK293T cell line. PVDF membrane was probed with 1.0 ug/ml of Rabbit Anti-Human HMGB1 Polyclonal Antibody (Catalog # NB100-2322) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog #HAF008). Specific band was detected for HMGB1 at approximately 30 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in the knockout HEK293T cell line. This experiment was conducted under reducing conditions.
Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Total protein from SHSY-5Y, MCF7, Neuro2A and HeLa was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/mL anti-HMGB1 in 1% non-fat milk in TBST and detected with an anti-rabbit HRP secondary antibody using chemiluminescence.
Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322]

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322]

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322] - Staining of HMGB1 in mouse liver using NB100-2322.
Simple Western: HMGB1/HMG-1 Antibody [NB100-2322]

Simple Western: HMGB1/HMG-1 Antibody [NB100-2322]

Simple Western: HMGB1/HMG-1 Antibody [NB100-2322] - Image shows a specific band for HMGB1 in 0.05 mg/mL of Jurkat lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - MCF7 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-HMGB1/HMG-1 Antibody NB100-2322 at 1 ug/ml for overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - Neuro2a cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-HMGB1 at 5 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322]

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322]

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322] - Rabbit blood vessel. Image from verified customer review.
Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

HMGB1-HMG-1-Antibody-Western-Blot-NB100-2322-img0026.jpg
Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-HMGB1/HMG-1 Antibody NB100-2322 at 1 ug/ml for overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322]

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322]

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on HeLa cells with HMGB1/HMG-1 Antibody NB100-2322AF488 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 488.
Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322]

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Hepatocyte protein lysate at 1:1000 4C overnight. Image from verfified customer review.
Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/ Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322]

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-HMGB1 [NB100-2322] at a 1:200 dilution overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) [NB100-690] was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Flow (Intracellular): HMGB1/HMG-1 Antibody [NB100-2322]

Flow (Intracellular): HMGB1/HMG-1 Antibody [NB100-2322]

Flow (Intracellular): HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on HeLa with NB100-2322 and a matched isotype control. Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody.
Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322]

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322]

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on RH-30 cells with NB100-2322F (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to FITC.
ELISA: HMGB1/HMG-1 Antibody [NB100-2322]

ELISA: HMGB1/HMG-1 Antibody [NB100-2322]

ELISA: HMGB1/HMG-1 Antibody [NB100-2322] - A dose-dependent titration of the HRP conjugated anti-HMGB1 antibody on recombinant human HMGB1 protein. Image from verified customer review. Image using the HRP format of this antibody.
HMGB1/HMG-1 Antibody

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] -

Expression of endogenous ligands for TLR2. (A) Expression of biglycan in AD-TLR2KO mice increased significantly compared with that in WT, AD, and TLR2KO mice (p<0.05). (B) HMGB1 in the four groups did not show a significant difference (p>0.05).
HMGB1/HMG-1 Antibody

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] -

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Expression of endogenous ligands for TLR2. (A) Expression of biglycan in AD-TLR2KO mice increased significantly compared with that in WT, AD, & TLR2KO mice (p<0.05). (B) HMGB1 in the four groups did not show a significant difference (p>0.05). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31509519), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for HMGB1/HMG-1 Antibody - BSA Free

Application
Recommended Usage

Flow Cytometry

1:100

Immunocytochemistry/ Immunofluorescence

0.05 ug/ml

Immunohistochemistry

1:100-1:250

Immunohistochemistry-Paraffin

1:100-1:250

Simple Western

1:2000

Western Blot

0.5-1.0 ug/ml
Application Notes
In Western blot a band is seen approx. 29 kDa. It has been reported that the HRP conjugated format (Catalog# NB100-2322H) of this antibody works well for use in ELISA.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in Jurkat lysate 0.05 mg/mL, separated by Size, antibody dilution of 1:2000. Separated by Size-Wes, Sally Sue/Peggy Sue.
The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reviewed Applications

Read 5 reviews rated 4.4 using NB100-2322 in the following applications:

Flow Cytometry Panel Builder

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C. Do not freeze.

Background: HMGB1/HMG-1

HMGB1 (high mobility group protein B1) is a member of HMGB family and it has been extensively studied as a transcription/growth factor that is most ubiquitous and evolutionarily conserved protein in eukaryotic organisms. HMGB1 is localized in nucleus, however gets translocated to cytoplasm or may get secreted also. It is a DNA binding protein that preferentially binds with ssDNA and contains 2 HMG box DNA-binding domains with ability of chromatin association for bending DNA. HMGB1 implicates in in V(D)J recombination by acting as a cofactor of RAG complex that contains core components RAG1 and RAG2, as well as an associated component HMGB1 or HMGB2. HMGB1 acts by stimulating cleavage and RAG protein binding at 23bp spacer of conserved RSS (recombination signal sequences). Originally described as a nonhistone nuclear protein with transcriptional regulatory properties, HMGB1 (especially the cytoplasmic/secreted form) has also now been recognized as a late mediator in septic shock as well as a pro-inflammatory cytokine. It gets translocated to cytoplasm or secreted when cells get exposed to inflammatory mediators (LPS, TNF-alpha, IFN-gamma) or via passive process during cellular necrosis. HMGB1 has been implicated in the pathogenesis of various inflammatory and autoimmune diseases, such as SLE, RA and ankylosing spondylitis.

Long Name

High Mobility Group Protein 1

Alternate Names

HMG-1, HMG1

Gene Symbol

HMGB1

Additional HMGB1/HMG-1 Products

Product Documents for HMGB1/HMG-1 Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for HMGB1/HMG-1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for HMGB1/HMG-1 Antibody - BSA Free

Customer Reviews for HMGB1/HMG-1 Antibody - BSA Free (5)

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Customer Images

Showing  1 - 5 of 5 reviews Showing All
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  • HMGB1/HMG-1 Antibody
    Name: seulgee lee
    Application: Immunohistochemistry-Paraffin
    Sample Tested: veesel
    Species: Rabbit
    Verified Customer | Posted 04/24/2018
    HMGB1/HMG-1 Antibody - BSA Free NB100-2322
  • HMGB1/HMG-1 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Brain homogenate
    Species: Rat
    Verified Customer | Posted 03/28/2018
    Lane 1: protein ladder. Lanes 2-7 decreasing protein load from 30 ug to 5 ug protein per well. Upper bands are B-actin, lower bands are HMGB1. The HMGB1 antibody exhibited excellent linearity with an r2 of 0.959.
    Blocked in 5% milk + 1% BSA in TBS for 2 h, incubated overnight in 1:500 HMGB1 antibody at 4 degrees C and then 2 h at room temperature with HRP-conjugated secondary.
    HMGB1/HMG-1 Antibody - BSA Free NB100-2322
  • HMGB1/HMG-1 Antibody
    Name: Liyong Zhang
    Application: Western Blot
    Sample Tested: mouse hepatocytes and HepG2 cells
    Species: Human
    Verified Customer | Posted 11/25/2017
    HMGB1/HMG-1 Antibody - BSA Free NB100-2322
  • Name: Anonymous
    Application: Simple Western
    Sample Tested: mouse serum
    Species: Mouse
    Verified Customer | Posted 12/30/2015
    HMGB1 in mouse serum protein extract
    HMGB1/HMG-1 Antibody - BSA Free NB100-2322
  • Name: Liyong Zhang
    Application: Western Blot
    Sample Tested:
    Species: Mouse
    Verified Customer | Posted 06/26/2014
    HMGB1/HMG-1 Antibody - BSA Free NB100-2322

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Protocols

View specific protocols for HMGB1/HMG-1 Antibody - BSA Free (NB100-2322):


Immunohistochemistry-paraffin embedded sections

Antigen Unmasking
Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.

Staining
1. Wash sections in dH2O three times for 5 minutes each.
2. Wash section in wash buffer (1X PBS/0.1% Tween-20 (1X PBST)) for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1X PBST, 5% goat serum) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul primary antibody diluted in 1X PBST, 5% goat serum to each section. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated secondary antibody, diluted in 1X PBST, 5% goat serum. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Striptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in dH2O.
12. Counterstain sections in hematoxylin.
13. Wash sections in dH2O two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.


Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.

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FAQs for HMGB1/HMG-1 Antibody - BSA Free

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  • Q: I'd like to use NB100-2322 for FACS. One of the images shows successful staining. Which permeabilization method and fixative was used? I've seen conflicting reports, especially for fixation.

    A: This image was obtained using 10% formalin as fixative and 90% methanol for permeabilization.

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