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Mouse Pyruvate kinase isozymes M1/M2 (Pkm2) ELISA Kit (MOEB0474)

SKU:
MOEB0474
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P52480
ELISA Type:
Sandwich
Synonyms:
M2-PK, CTHBP, OIP3
Reactivity:
Mouse
€649
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Description

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Mouse Pyruvate kinase isozymes M1/M2 (Pkm2) ELISA Kit

The Mouse Pyruvate Kinase Isozymes M1/M2 (PKM2) ELISA Kit is a powerful tool for accurately measuring levels of PKM2 in mouse serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers reliable and reproducible results, making it an excellent choice for a variety of research purposes.PKM2 is an important enzyme involved in glycolysis and plays a crucial role in regulating cellular metabolism. Dysregulation of PKM2 has been linked to various diseases, including cancer and metabolic disorders, making it a valuable biomarker for understanding disease mechanisms and developing potential therapeutic interventions.

Overall, the Mouse Pyruvate Kinase Isozymes M1/M2 (PKM2) ELISA Kit offers researchers a reliable and accurate means of studying PKM2 levels in mouse samples, providing valuable insights into the role of this enzyme in disease pathology and potential treatment strategies.

Product Name:Mouse Pyruvate kinase isozymes M1/M2 (Pkm2) ELISA Kit
SKU:MOEB0474
Size:96T
Target:Mouse Pyruvate kinase isozymes M1/M2 (Pkm2)
Synonyms:Pyruvate kinase muscle isozyme, Pk3, Pkm2, Pykm
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:0.78-50U/mL
Sensitivity:0.38U/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP. Stimulates POU5F1-mediated transcriptional activation.
Uniprot:P52480
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse Pyruvate kinase PKM
Sub Unit:Monomer and homotetramer. Exists as a monomer in the absence of fructose 1,6 bi-phosphate (FBP), and reversibly associates to form a homotetramer in the presence of FBP. The monomeric form binds T3. Tetramer formation induces pyruvate kinase activity. FBP stimulates the formation of tetramers from dimers. Interacts with HERC1, POU5F1 and PML. Interacts (isoform M2) with EGLN3; the interaction hydroxylates PKM under hypoxia and enhances binding to HIF1A. Interacts (isoform M2) with HIF1A; the interaction is enhanced by binding of EGLN3, promoting enhanced transcription activity under hypoxia (By similarity). Interacts (isoform M2, but not isoform M1) with TRIM35; this interaction prevents FGFR1-dependent tyrosine phosphorylation.
Research Area:Epigenetics
Subcellular Location:Cytoplasm Nucleus Translocates to the nucleus in response to different apoptotic stimuli. Nuclear translocation is sufficient to induce cell death that is caspase independent, isoform-specific and independent of its enzymatic activity.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP. Stimulates POU5F1-mediated transcriptional activation.
UniProt Code:P52480
NCBI GenInfo Identifier:146345448
NCBI Gene ID:18746
NCBI Accession:P52480.4
UniProt Secondary Accession:P52480,Q3TBV8, Q3TBW5, Q3TC59, Q3U1X3, Q3U5P6, Q4VC20 Q64484, Q91YI8, Q9CWB1,
UniProt Related Accession:P52480
Molecular Weight:57,985 Da
NCBI Full Name:Pyruvate kinase PKM
NCBI Synonym Full Names:pyruvate kinase, muscle
NCBI Official Symbol:Pkm
NCBI Official Synonym Symbols:Pk3; Pk-2; Pk-3; Pkm2; AA414905; AL024370; AL024424
NCBI Protein Information:pyruvate kinase PKM
UniProt Protein Name:Pyruvate kinase PKM
UniProt Synonym Protein Names:Pyruvate kinase muscle isozyme
UniProt Gene Name:Pkm
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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