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.

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

Cited:

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

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

1 mg/ml

View all HMGB1/HMG-1 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

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

Knockdown Validated: 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] - 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] - 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] - 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] - 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] - 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] - Rabbit blood vessel. Image from verified customer review.

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

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - The onset of innate immune response after I/R injury was compared in wt and PT-SSAT-Cko animals. Time course of the expression of HMGB1, TLR2 and 4 were compared in the kidneys of sham-operated and injured wt and PT-SSAT-Cko mice. The data are representative of three independent experiments. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0110161) licensed under a CC-BY license.

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] - 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] - 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] - 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] - 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] - 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] - 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.

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).

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.

View 14 more images

Applications for HMGB1/HMG-1 Antibody

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. 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:

Immunohistochemistry-Paraffin (1 Review)
Simple Western (1 Review)
Western Blot (3 Reviews)

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

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

Product Datasheets

Download Product Datasheets

Protocols & Manuals

Download IHC-P Protocol

Download Western Blot Protocol

COA

SDS

Download SDS

Product Specific Notices for HMGB1/HMG-1 Antibody

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
Reviews

Protocols
FAQs

HMGB1/HMG-1 Antibody

Novus Biologicals, part of Bio-Techne | Catalog # NB100-2322

Key Product Details

Validated by

Species Reactivity

Validated:

Cited:

Predicted:

Applications

Validated:

Cited:

Label

Antibody Source

Concentration

View all HMGB1/HMG-1 Antibodies »

Product Specifications

Immunogen

Reactivity Notes

Localization

Clonality

Host

Isotype

Theoretical MW

Scientific Data Images for HMGB1/HMG-1 Antibody

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Applications for HMGB1/HMG-1 Antibody

Flow Cytometry

Immunocytochemistry/ Immunofluorescence

Immunohistochemistry

Immunohistochemistry-Paraffin

Simple Western

Western Blot

Reviewed Applications

Formulation, Preparation, and Storage

Purification

Formulation

Preservative

Concentration

Shipping

Stability & Storage

Background: HMGB1/HMG-1

Long Name

Alternate Names

Gene Symbol

Additional HMGB1/HMG-1 Products

Product Documents for HMGB1/HMG-1 Antibody

Product Datasheets

Protocols & Manuals

COA

Certificate of Analysis

SDS

Product Specific Notices for HMGB1/HMG-1 Antibody