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Porcine High mobility group protein B2 (HMGB2) ELISA Kit (PREB0399)

SKU:
PREB0399
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P17741
ELISA Type:
Sandwich
Reactivity:
Porcine
$839
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Description

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Porcine High mobility group protein B2 (HMGB2) ELISA Kit

The Porcine High Mobility Group Protein B2 (HMGB2) ELISA Kit is specially designed for the accurate measurement of HMGB2 levels in porcine samples. This kit offers high sensitivity and specificity, allowing for reliable and reproducible results that are ideal for various research applications.HMGB2 is a protein that belongs to the high mobility group (HMG) family and is known for its involvement in DNA bending, transcription regulation, and chromatin remodeling. It plays a crucial role in various cellular processes such as DNA repair, inflammation, and immune response.

The measurement of HMGB2 levels can provide valuable insights into the pathogenesis of diseases such as cancer, inflammatory disorders, and autoimmune diseases.Overall, the Porcine HMGB2 ELISA Kit is an essential tool for researchers studying the role of HMGB2 in different biological processes and disease conditions, allowing for a better understanding of its function and potential therapeutic applications.

Product Name:Porcine High mobility group protein B2 (HMGB2) ELISA Kit
SKU:PREB0399
Size:96T
Target:Porcine High mobility group protein B2 (HMGB2)
Synonyms:High mobility group protein 2, HMG-2, HMG2
Detection Method:ELISA
Reactivity:Pig
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
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 (PubMed:11275566). 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:P17741
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant pig 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-regulatorCellular Component: cell; condensed chromosome; cytoplasm; extracellular space; nuclear chromatin; nucleus; perinuclear region of cytoplasmMolecular Function: DNA binding; DNA binding, bending; double-stranded DNA binding; drug binding; four-way junction DNA binding; protein domain specific binding; single-stranded DNA binding; transcription factor bindingBiological Process: chemotaxis; defense response to Gram-negative bacterium; defense response to Gram-positive bacterium; DNA geometric change; DNA ligation involved in DNA repair; DNA recombination; DNA topological change; inflammatory response to antigenic stimulus; innate immune response; male gonad development; nucleosome assembly; positive regulation of DNA binding; positive regulation of transcription from RNA polymerase II promoter; regulation of neurogenesis; regulation of transcription from RNA polymerase II promoter; response to drug; response to lipopolysaccharide; response to steroid hormone; spermatid nuclear differentiation; spermatogenesis; transcription, DNA-dependent
UniProt Protein Details:
NCBI Summary:
UniProt Code:P17741
NCBI GenInfo Identifier:47522942
NCBI Gene ID:397130
NCBI Accession:NP_999228.1
UniProt Secondary Accession:P17741
UniProt Related Accession:P17741
Molecular Weight:24,133 Da
NCBI Full Name:high mobility group protein B2
NCBI Synonym Full Names:
NCBI Official Symbol:HMGB2
NCBI Official Synonym Symbols:HMG2; HMG-2
NCBI Protein Information:high mobility group protein B2
UniProt Protein Name:High mobility group protein B2
UniProt Synonym Protein Names:High mobility group protein 2; HMG-2
Protein Family:High mobility group protein
UniProt Gene Name:HMGB2
UniProt Entry Name:
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 4°C
Plate Sealer 5 -

Other materials and equipment 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

*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.

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