Rat High mobility group protein B2 (Hmgb2) ELISA Kit
The Rat High Mobility Group Protein B2 (HMGB2) ELISA Kit is a powerful tool for accurately measuring HMGB2 levels in rat samples including serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures precise and consistent results, making it ideal for a wide range of research applications.HMGB2 is an important protein involved in DNA binding and repair processes, as well as gene transcription regulation.
It plays a critical role in various biological functions, making it a valuable biomarker for studying conditions such as inflammation, cancer, and autoimmune diseases. By utilizing this ELISA kit, researchers can gain valuable insights into HMGB2 levels and its implications in different diseases, ultimately leading to advancements in therapeutic strategies.
Product Name:
Rat High mobility group protein B2 (Hmgb2) ELISA Kit
SKU:
RTEB0991
Size:
96T
Target:
Rat High mobility group protein B2 (Hmgb2)
Synonyms:
High mobility group protein 2, HMG-2, Hmg2
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.156-10ng/mL
Sensitivity:
0.086ng/mL
Intra CV:
4.5%
Inter CV:
6.9%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
107-117%
103-115%
87-97%
82-82%
EDTA Plasma(N=5)
103-112%
104-113%
109-118%
105-118%
Heparin Plasma(N=5)
83-93%
110-120%
108-118%
90-99%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
97
91-103
Plasma
99
93-105
Function:
Multifunctional protein with various roles in different cellular compartments. May act in a redox sensitive manner. In the nucleus is an abundant chromatin-associated non-histone protein involved in transcription, chromatin remodeling and V(D)J recombination and probably other processes (By similarity). Binds DNA with a preference to non-canonical DNA structures such as single-stranded DNA. Can bent DNA and enhance DNA flexibility by looping thus providing a mechanism to promote activities on various gene promoters by enhancing transcription factor binding and/or bringing distant regulatory sequences into close proximity. Involved in V(D)J recombination by acting as a cofactor of the RAG complex: acts by stimulating cleavage and RAG protein binding at the 23 bp spacer of conserved recombination signal sequences (RSS). Proposed to be involved in the innate immune response to nucleic acids by acting as a cytoplasmic promiscuous immunogenic DNA/RNA sensor which cooperates with subsequent discriminative sensing by specific pattern recognition receptors. In the extracellular compartment acts as a chemokine. Promotes proliferation and migration of endothelial cells implicating AGER/RAGE. Has antimicrobial activity in gastrointestinal epithelial tissues. Involved in inflammatory response to antigenic stimulus coupled with proinflammatory activity. May play a role in germ cell differentiation. Involved in modulation of neurogenesis probably by regulation of neural stem proliferation. Involved in articular cartilage surface maintenance implicating LEF1 and the Wnt/beta-catenin pathway.
Uniprot:
P52925
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat High mobility group protein B2
Sub Unit:
Interacts with POU2F2, POU2F1 and POU3F1. Component of the RAG complex composed of core components RAG1 and RAG2, and associated component HMGB1 or HMGB2. Component of the SET complex, composed of at least ANP32A, APEX1, HMGB2, NME1, SET and TREX1. Directly interacts with SET. Interacts with LEF1.
Research Area:
Epigenetics
Subcellular Location:
Nucleus Chromosome Cytoplasm Secreted
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
HMGB2: DNA binding proteins that associates with chromatin and has the ability to bend DNA. Binds preferentially single-stranded DNA. Involved in V(D)J recombination by acting as a cofactor of the RAG complex. Acts by stimulating cleavage and RAG protein binding at the 23 bp spacer of conserved recombination signal sequences (RSS). Belongs to the HMGB family.Protein type: DNA-binding; Nuclear receptor co-regulatorChromosomal Location of Human Ortholog: 4q31Cellular Component: nucleoplasm; extracellular space; protein complex; perinuclear region of cytoplasm; cytoplasm; condensed chromosome; nucleusMolecular Function: protein domain specific binding; RAGE receptor binding; protein binding; DNA binding; double-stranded DNA binding; damaged DNA binding; chromatin binding; DNA bending activity; transcription factor activity; single-stranded DNA binding; chemoattractant activityBiological Process: positive regulation of nuclease activity; establishment and/or maintenance of chromatin architecture; DNA topological change; V(D)J recombination; apoptosis; positive regulation of transcription, DNA-dependent; positive regulation of erythrocyte differentiation; male gonad development; spermatid nuclear differentiation; base-excision repair, DNA ligation; regulation of transcription from RNA polymerase II promoter; chromatin remodeling; nucleosome assembly; positive chemotaxis; response to steroid hormone stimulus; DNA ligation during DNA repair; positive regulation of megakaryocyte differentiation; positive regulation of endothelial cell proliferation; positive regulation of transcription from RNA polymerase II promoter; DNA fragmentation during apoptosis; negative regulation of transcription, DNA-dependent; positive regulation of DNA binding; cell structure disassembly during apoptosis
UniProt Protein Details:
NCBI Summary:
This gene encodes a member of the non-histone chromosomal high mobility group protein family. The proteins of this family are chromatin-associated and ubiquitously distributed in the nucleus of higher eukaryotic cells. In vitro studies have demonstrated that this protein is able to efficiently bend DNA and form DNA circles. These studies suggest a role in facilitating cooperative interactions between cis-acting proteins by promoting DNA flexibility. This protein was also reported to be involved in the final ligation step in DNA end-joining processes of DNA double-strand breaks repair and V(D)J recombination. [provided by RefSeq, Jul 2008]
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
