Rat Glycogen synthase kinase-3 beta (Gsk3b) ELISA Kit
The Rat Glycogen Synthase Kinase-3 Beta (GSK3B) ELISA Kit is specifically designed for the quantitative measurement of GSK3B levels in rat serum, plasma, and cell lysates. This kit offers excellent sensitivity and specificity, ensuring accurate and reliable results for various research applications.GSK3B is a key enzyme involved in the regulation of numerous cellular processes, including cell growth, differentiation, and apoptosis. Dysregulation of GSK3B has been linked to various diseases, such as diabetes, Alzheimer's disease, and cancer, making it a valuable target for research and drug development.
By using the Rat GSK3B ELISA Kit, researchers can effectively study the role of GSK3B in different physiological and pathological states, thereby paving the way for potential therapeutic interventions. This kit provides a convenient and efficient tool for investigating the mechanisms underlying GSK3B-related disorders and identifying novel treatment strategies.
Product Name:
Rat Glycogen synthase kinase-3 beta (Gsk3b) ELISA Kit
SKU:
RTEB1479
Size:
96T
Target:
Rat Glycogen synthase kinase-3 beta (Gsk3b)
Synonyms:
Factor A, Serine/threonine-protein kinase GSK3B, FA, GSK-3 beta
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.312-20ng/mL
Sensitivity:
0.172ng/mL
Intra CV:
4.6%
Inter CV:
7.9%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
109-119%
94-104%
109-119%
104-113%
EDTA Plasma(N=5)
100-109%
86-98%
92-101%
102-111%
Heparin Plasma(N=5)
90-100%
103-113%
105-115%
84-97%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
94
88-100
Plasma
96
90-102
Function:
Constitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis, Wnt signaling and regulation of transcription factors and microtubules, by phosphorylating and inactivating glycogen synthase (GYS1 or GYS2), EIF2B, CTNNB1/beta-catenin, APC, AXIN1, DPYSL2/CRMP2, JUN, NFATC1/NFATC, MAPT/TAU and MACF1. Requires primed phosphorylation of the majority of its substrates. In skeletal muscle, contributes to insulin regulation of glycogen synthesis by phosphorylating and inhibiting GYS1 activity and hence glycogen synthesis. May also mediate the development of insulin resistance by regulating activation of transcription factors. Regulates protein synthesis by controlling the activity of initiation factor 2B (EIF2BE/EIF2B5) in the same manner as glycogen synthase. In Wnt signaling, GSK3B forms a multimeric complex with APC, AXIN1 and CTNNB1/beta-catenin and phosphorylates the N-terminus of CTNNB1 leading to its degradation mediated by ubiquitin/proteasomes. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA. Phosphorylates NFATC1/NFATC on conserved serine residues promoting NFATC1/NFATC nuclear export, shutting off NFATC1/NFATC gene regulation, and thereby opposing the action of calcineurin. Phosphorylates MAPT/TAU on 'Thr-548', decreasing significantly MAPT/TAU ability to bind and stabilize microtubules. Plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. Phosphorylates MACF1, inhibiting its binding to microtubules which is critical for its role in bulge stem cell migration and skin wound repair. Probably regulates NF-kappa-B (NFKB1) at the transcriptional level and is required for the NF-kappa-B-mediated anti-apoptotic response to TNF-alpha (TNF/TNFA). Negatively regulates replication in pancreatic beta-cells, resulting in apoptosis, loss of beta-cells. Through phosphorylation of the anti-apoptotic protein MCL1, may control cell apoptosis in response to growth factors deprivation. Phosphorylates MUC1 in breast cancer cells, decreasing the interaction of MUC1 with CTNNB1/beta-catenin. Is necessary for the establishment of neuronal polarity and axon outgrowth. Phosphorylates MARK2, leading to inhibit its activity. Phosphorylates SIK1 at 'Thr-182', leading to sustain its activity. Phosphorylates ZC3HAV1 which enhances its antiviral activity. Phosphorylates SFPQ upon T-cell activation. Phosphorylates SNAI1, leading to its BTRC-triggered ubiquitination and proteasomal degradation. Phosphorylates NR1D1 st 'Ser-55' and 'Ser-59' and stabilizes it by protecting it from proteasomal degradation. Regulates the circadian clock via phosphorylation of the major clock components including ARNTL/BMAL1, CLOCK and PER2. Phosphorylates CLOCK AT 'Ser-427' and targets it for proteasomal degradation. Phosphorylates ARNTL/BMAL1 at 'Ser-17' and 'Ser-21' and primes it for ubiquitination and proteasomal degradation. Phosphorylates OGT at 'Ser-3' or 'Ser-4' which positively regulates its activity.
Uniprot:
P18266
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Glycogen synthase kinase-3 beta
Sub Unit:
Monomer. Interacts with DAB2IP (via C2 domain); the interaction stimulates GSK3B kinase activation. Interacts (via C2 domain) with PPP2CA (By similarity). Interacts with ARRB2, AXIN1, CABYR, DISC1, MMP2, MUC1, NIN, PRUNE and ZBED3 (By similarity). Interacts with AXIN1; the interaction mediates hyperphosphorylation of CTNNB1 leading to its ubiquitination and destruction. Interacts with and phosphorylates SNAI1. Interacts with DNM1L (via a C-terminal domain) (By similarity). Found in a complex composed of MACF1, APC, AXIN1, CTNNB1 and GSK3B. Interacts with SGK3. Interacts with the CLOCK-ARNTL/BMAL1 heterodimer. Interacts with the ARNTL/BMAL1 (By similarity). Interacts with CTNND2.
Research Area:
Cancer
Subcellular Location:
Cytoplasm Nucleus Membrane Cell membrane The phosphorylated form shows localization to cytoplasm and cell membrane. The MEMO1-RHOA-DIAPH1 signaling pathway controls localization of the phosphorylated form to the cell membrane (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
GSK3B: a proline-directed protein kinase of the GSK family. Phosphorylates and inactivates glycogen synthase. Participates in the Wnt signaling pathway. Involved in energy metabolism, neuronal cell development, and body pattern formationProtein type: CMGC group; EC 2.7.11.1; EC 2.7.11.26; GSK family; GSK subfamily; Kinase, protein; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor)Chromosomal Location of Human Ortholog: 11q21Cellular Component: axon; beta-catenin destruction complex; cell soma; centrosome; cytoplasm; cytosol; dendrite; dendritic shaft; dendritic spine; growth cone; lipid raft; membrane; microtubule; mitochondrion; nucleus; perinuclear region of cytoplasm; plasma membrane; postsynaptic density; protein complex; ribonucleoprotein complexMolecular Function: ATP binding; beta-catenin binding; integrin binding; ionotropic glutamate receptor binding; kinase activity; NF-kappaB binding; p53 binding; protease binding; protein binding; protein kinase activity; protein kinase binding; protein serine/threonine kinase activity; tau protein binding; tau-protein kinase activity; transcription factor binding; ubiquitin protein ligase bindingBiological Process: aging; axonogenesis; cell migration; circadian rhythm; epithelial to mesenchymal transition; ER overload response; establishment and/or maintenance of cell polarity; establishment of cell polarity; fat cell differentiation; genetic imprinting; glycogen metabolic process; hippocampus development; insulin receptor signaling pathway; myoblast fusion; myotube differentiation; negative regulation of apoptosis; negative regulation of dendrite morphogenesis; negative regulation of MAP kinase activity; negative regulation of neuron maturation; negative regulation of NFAT protein import into nucleus; negative regulation of nitric-oxide synthase activity; negative regulation of protein binding; negative regulation of protein complex assembly; negative regulation of TOR signaling pathway; organ morphogenesis; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; phosphorylation; positive regulation of apoptosis; positive regulation of autophagy; positive regulation of axon extension; positive regulation of cell-matrix adhesion; positive regulation of GTPase activity; positive regulation of neuron apoptosis; positive regulation of peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein binding; positive regulation of protein catabolic process; positive regulation of protein complex assembly; positive regulation of protein export from nucleus; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein export from nucleus; re-entry into mitotic cell cycle; regulation of axon extension; regulation of axonogenesis; regulation of dendrite morphogenesis; regulation of microtubule-based process; regulation of neuronal synaptic plasticity; response to activity; response to drug; response to estradiol stimulus; response to insulin stimulus; response to lithium ion; Wnt receptor signaling pathway; Wnt receptor signaling pathway through beta-catenin
UniProt Protein Details:
NCBI Summary:
mediates Par6-atypical protein kinase C (aPKC) complex regulation of cell polarity; may induce apoptosis [RGD, Feb 2006]
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.
