Hmgb1 primary antibody

Manufactured by Abcam

HMGB1 primary antibody is a laboratory reagent used for the detection and identification of HMGB1 (High Mobility Group Box 1) protein in various samples. HMGB1 is a highly conserved nuclear protein involved in the regulation of gene transcription. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of HMGB1 in biological samples.

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Lab products found in correlation

Horse anti mouse hrp conjugate, by Cell Signaling Technology (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions) Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions) Alexa fluor 680 goat anti rabbit igg, by LI COR (1 mentions) Odyssey clx imager, by LI COR (1 mentions) Alexa fluor 488 donkey anti rabbit igg, by Abcam (1 mentions) U tbi90, by Olympus (1 mentions) Confocal microscope, by Olympus (1 mentions) Supersignal west femto, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions) Quantity one, by Bio-Rad (1 mentions)

Close spelling variants of this product

HMGB1 (192 citations) HMGB1 antibody (23 citations) Anti-HMGB1 primary antibody (2 citations)

4 protocols using hmgb1 primary antibody

Western Blot Immunodetection of HMGB1, RIPK3, and Bak

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Twenty‐five microliters of cell‐free supernatant or 25 mg of total protein cell lysate was resolved on a 4–12% NUPAGE™ pre‐cast SDS‐polyacrylamide gel electrophoresis (PAGE) gel (Life Technologies Inc.) at 90 V (15 min) then 180 V (60 min). Transfer of immobilized proteins to nitrocellulose membrane was carried out at 100 V for 1 h and the membrane was blocked in Tris‐buffered saline‐tween containing 10% dried‐milk powder for 1 h at room temperature. Membranes were probed with rabbit anti‐human high‐mobility group box 1 (HMGB1) primary antibody (1:5000 dilution, overnight; Abcam Ltd) and goat anti‐rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)‐conjugate secondary antibody (1:10 000 dilution, 2 h; Sigma‐Aldrich).
Streptavidin‐tagged RIPK3 and Bak were probed with strepMAB‐classic mouse anti‐human antibody (1:4000; IBA Life Sciences) and horse anti‐mouse HRP‐conjugate (1:10 000 dilution; Cell Signaling Technologies). Details of all other primary and secondary antibodies used are given in Supporting Information Table S3. Membranes were developed and visualized using enhanced chemiluminescent substrate (ECL) and a Chemidoc Touch Imaging System (both Bio‐Rad). Image densitometric analysis was undertaken using Image J software.

Nwikue G., Olsson‐Brown A., Aboheimed N., Yip V., Jolly C., Luchian A., Ressel L., Sharma A., Bergfeld W., Ahmed S., Dickinson A., Pirmohamed M, & Carr D.F. (2023). TNF‐α induced extracellular release of keratinocyte high‐mobility group box 1 in Stevens‐Johnson syndrome/toxic epidermal necrolysis: Biomarker and putative mechanism of pathogenesis. The Journal of Dermatology, 50(9), 1129-1139.

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HMGB1 Protein Detection in BALF

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Equal volumes of BALF were subjected to electrophoresis on NuPAGE 4-12% Bis-Tris gradient gel (Life Technologies, Carlsbad, CA), and transferred to PVDF membrane by using iBlot™ gel transfer device (Life Technologies, Carlsbad, CA). Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA). Alexa Fluor 680 goat anti-rabbit IgG (926-68071; LI-COR Biosciences, Lincoln, NE) was used as the secondary antibody. Protein bands were visualized using Odyssey CLx, Imager (LI-COR Biosciences, Lincoln, NE).

Mao Y., Patial S, & Saini Y. (2023). Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease. Frontiers in Immunology, 13, 944772.

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Immunofluorescence Analysis of HMGB1 in Glioma and Astrocyte Cells

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The glioma and astrocyte cell lines were cultured over sterile glass coverslips and then fixed for 20 min using: i) 4% PFA at RT; or ii) methanol with 5% (v/v) acetic acid at −20°C. The cells were then rehydrated and permeabilized using 0.5% Triton X-100 (in PBS) for 5 min at RT, prior to being blocked with 3% BSA for 1 h at RT. The cells were then incubated with a HMGB1 primary antibody (1:1,000 diluted in 1% BSA; Abcam; cat. no. ab18256) overnight at 4°C, followed by subsequent incubation with Alexa Fluor 488 donkey anti-rabbit IgG (1:1,000; Abcam; cat. no. ab150073). The cells were then counterstained using DAPI. Coverslips were mounted in 50% (v/v) glycerol with PBS, and observed under an Olympus confocal microscope (magnification, ×100; cat. no. U-TBI90; Olympus Corporation).

Jia L., Song H., Fan W., Song Y., Wang G., Li X., He Y, & Yao A. (2019). The association between high mobility group box 1 chromatin protein and mitotic chromosomes in glioma cells. Oncology Letters, 19(1), 745-752.

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Quantification of HMGB-1 in Vitreous Humor

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To collect vitreous humor (VH), the sclera was penetrated by using a 33G needle at a point temporal and posterior to the limbus, behind the lens. A small drop of VH that drained out was collected. This collected volume was equal to 2.5 µl, which was transferred to the tube containing 12.5 µl of PBS. The tube was centrifuged at 1100 RPM for 15 minutes at +4°C and the supernatant was collected. To evaluate the HMGB-1 level, 10 µl of the supernatant were loaded, and the proteins were size-separated in SDS PAGE gel. Proteins were blotted onto a PVDF membrane (Invitrogen, Carlsbad, CA) and incubated with HMGB-1 primary antibody (1∶1000, Abcam, Cambridge, MA). Proteins recognized by the antibody were revealed by an ultrasensitive chemiluminescent substrate system (SuperSignal West Femto, Thermo Scientific, Rockford, IL) according to instructions. Quantification of the protein bands was performed using image acquisition and analysis software (Quantity One; Bio-Rad Laboratories, Hercules, CA).

Santos A.R., Dvoriantchikova G., Li Y., Mohammad G., Abu El-Asrar A.M., Wen R, & Ivanov D. (2014). Cellular Mechanisms of High Mobility Group 1 (HMGB-1) Protein Action in the Diabetic Retinopathy. PLoS ONE, 9(1), e87574.

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