Human HMGB1 ELISA Kit (HUFI00660)

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human HMGB1 ELISA Kit
Product Code:	HUFI00660
Size:	96 Assays
Alias:	HMGB1, High mobility group protein B1, HMG-1, HMG1, HMG3, SBP-1, Amphoterin, high mobility group box 1, High mobility group protein 1, high-mobility group, nonhistone chromosomal protein 1, high-mobility group box 1, Sulfoglucuronyl carbohydRate binding protein
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This ELISA kit allows for the in vitro quantitative determination of Human HMGB1 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human HMGB1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human HMGB1 in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	87-102	91
EDTA plasma(n=5)	90-96	92
UFH plasma(n=5)	90-103	98

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HMGB1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
serum(n=5)	85-104%	90-103%	86-105%
EDTA plamsa(n=5)	83-98%	82-97%	84-92%
UFH plasma(n=5)	81-96%	82-97%	83-96%

CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protein Information

Heading 1	Heading 2
UniProt Code	P09429
NCBI GenInfo Identifier	123369
NCBI Gene ID	3146
NCBI Accession	P09429.3
Molecular Weight	24,894 Da

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Type

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

HMGB1 Background

HMGB1

The non-histone chromosome-binding protein known as the high mobility group (HMG) is named after its low molecular weight and great gel mobility in eukaryotic cells. HMG proteins are grouped into three gene families: HMGA, HMGB, and HMGN based on the molecular weight, structural similarity, and DNA binding properties. The most prevalent nonhistone nucleoprotein in the HMGB gene family is known as HMGB1, often referred to as amphoterin or HMG1. Due to its constant movement between the nucleus and the cytoplasm, HMGB1 is also expressed there to some extent. Nonhistone nucleoprotein HMGB1 serves as both an external inflammatory cytokine and a nonhistone nucleoprotein. Involved in transcriptional control, DNA replication and repair, telomere preservation, and nucleosome assembly, intracellular HMGB1 is strongly linked to DNA. Stressed or necrotic cells release extracellular HMGB1 either passively or aggressively. It functions as a damage-associated molecular pattern (DAMP) by binding to pattern recognition receptors (PRRs) as a chemokine or cytokine.

HMGB1 Structure

The HMGB1 gene is located on chromosome 13q12 and includes five exons and four introns. HMGB1, or High Mobility Group Box 1, is a highly conserved protein found in the nucleus of cells. It consists of 215 amino acids and is characterized by two DNA-binding domains known as Box A and Box B. HMGB1 plays a crucial role in various cellular processes, including DNA repair, transcription regulation, and inflammation. Its unique structure allows it to interact with DNA, as well as other proteins, contributing to its diverse functions. Understanding the structure of HMGB1 is essential for unraveling its intricate mechanisms and exploring its potential applications in research and therapeutics

HMGB1 Role and Function

HMGB1, also known as High Mobility Group Box 1, is a protein that plays a crucial role in various biological processes within the body. Acting as a potent pro-inflammatory molecule, HMGB1 is released by immune cells during infection, injury, or tissue damage. It acts as a danger signal, alerting the immune system to initiate a response against potential threats. Additionally, HMGB1 is involved in tissue regeneration and repair, promoting cell migration and proliferation.

HMGB1 ELISA Kit FAQs

What is the HMGB1 ELISA Kit used for?

The Human HMGB1 ELISA Kit is specifically designed for the quantitative measurement of High Mobility Group Box 1 (HMGB1) protein levels in human samples. HMGB1 is a highly conserved nuclear protein that can be released by activated immune cells or damaged tissues, playing a crucial role in inflammation, immune response, and various disease processes. This ELISA kit enables researchers to accurately detect and quantify HMGB1 levels, facilitating investigations into its role in disease pathogenesis and therapeutic interventions.

What are the advantages of using HMGB1 ELISA Kit?

The Human HMGB1 ELISA Kit offers several advantages. It provides a highly sensitive and specific method for the quantification of HMGB1 levels in human samples, ensuring accurate and reliable results. The kit offers a user-friendly protocol with clear instructions, making it suitable for both experienced researchers and beginners. With its optimized reagents and standardized procedures, the kit offers consistent and reproducible results, minimizing experimental variations. Furthermore, the kit provides a rapid and cost-effective solution for HMGB1 analysis, saving valuable time and resources in the laboratory.

What sample types are compatible with HMGB1 ELISA kit?

The HMGB1 ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze HMGB1 levels in different biological matrices.

What are the storage requirements for the HMGB1 ELISA Kit?

The HMGB1 ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide riubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the HMGB1 ELISA kit.

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Human HMGB1 ELISA Kit

Description