Rat Integrin-linked protein kinase (Ilk) ELISA Kit
The Rat Integrin-Linked Protein Kinase (ILK) ELISA Kit is a comprehensive tool for the accurate measurement of ILK levels in rat samples, including serum, plasma, and cell culture supernatants. This kit boasts exceptional sensitivity and specificity, guaranteeing precise and reproducible results that are suitable for a diverse range of scientific investigations.ILK is a key enzyme that functions in cell signaling pathways, playing a critical role in cell adhesion, migration, and survival. Dysregulation of ILK is implicated in various diseases, including cancer, cardiovascular disorders, and muscle diseases.
As such, measuring ILK levels can provide valuable insights into disease progression and potential therapeutic strategies.With the Rat Integrin-Linked Protein Kinase (ILK) ELISA Kit, researchers can explore the mechanistic roles of ILK in disease pathogenesis and assess its potential as a therapeutic target. This kit offers a reliable and efficient solution for studying ILK biology and its implications in various disease conditions.
Product Name:
Rat Integrin-linked protein kinase (Ilk) ELISA Kit
SKU:
RTEB1112
Size:
96T
Target:
Rat Integrin-linked protein kinase (Ilk)
Synonyms:
Integrin-linked protein kinase, Ilk, 2.7.11.1
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.156-10ng/ml
Sensitivity:
0.078ng/mL
Intra CV:
4.5%
Inter CV:
8.2%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
116-126%
90-99%
110-120%
97-107%
EDTA Plasma(N=5)
100-110%
84-95%
102-112%
103-116%
Heparin Plasma(N=5)
97-107%
90-100%
110-120%
84-94%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
101
95-107
Plasma
103
97-109
Function:
Receptor-proximal protein kinase regulating integrin-mediated signal transduction. May act as a mediator of inside-out integrin signaling. Focal adhesion protein part of the complex ILK-PINCH. This complex is considered to be one of the convergence points of integrin- and growth factor-signaling pathway. Could be implicated in mediating cell architecture, adhesion to integrin substrates and anchorage-dependent growth in epithelial cells. Phosphorylates beta-1 and beta-3 integrin subunit on serine and threonine residues, but also AKT1 and GSK3B.
Uniprot:
Q99J82
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Integrin-linked protein kinase
Sub Unit:
Interacts with FERMT2 (By similarity). Interacts with the cytoplasmic domain of ITGB1. Could also interact with integrin ITGB2, ITGB3 and/or ITGB5. Interacts (via ANK repeats) with LIMS1 and LIMS2. Interacts with PARVA and PARVB; these compete for the same binding site. Interacts probably also with TGFB1I1. Interacts (via ANK repeats) with EPHA1 (via SAM domain); stimulated by EFNA1 but independent of the kinase activity of EPHA1. Interacts with LIMD2; leading to activate the protein kinase activity.
Research Area:
Cancer
Subcellular Location:
Cell junction Focal adhesion Cell membrane Peripheral membrane protein Cytoplasmic side Cell projection Lamellipodium Cytoplasm Myofibril Sarcomere
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
ILK: a tyrosine kinase-like kinase of the MLK family. Couples integrins and growth factors to downstream pathways involved in cell survival, cell cycle control, cell-cell adhesion and cell motility. Functions as a scaffold bridging the extra-cellular matrix and growth factor receptors to the actin cytoskeleton through interactions with the ILK-PINCH complex. This complex, which includes integrin, PINCH (which links ILK to receptor tyrosine kinases via Nck2), PARVA and affixin, is considered to be one of the convergence points of integrin- and growth factor-signaling pathways. May be implicated in mediating cell architecture, adhesion to integrin substrates and anchorage-dependent growth in epithelial cells. Stimulated downstream of PI3 kinase. Increased expression is correlated with progression of several tumor types, including breast, prostate, and colon carcinomas. Overexpression drives anchorage-independent growth and faster cell cycle.Protein type: EC 2.7.11.1; Kinase, protein; Protein kinase, TKL; Protein kinase, Ser/Thr (non-receptor); TKL group; MLK family; ILK subfamilyChromosomal Location of Human Ortholog: 11p15.4Cellular Component: protein complex; costamere; focal adhesion; terminal button; intercellular junction; cytosol; nucleoplasm; sarcomere; membrane; cell soma; lamellipodium; cytoplasm; plasma membrane; stress fiber; dendritic shaft; cell junctionMolecular Function: integrin binding; protein serine/threonine kinase activity; protein binding; signal transducer activity; SH3 domain binding; protein kinase binding; ATP bindingBiological Process: negative regulation of smooth muscle cell proliferation; protein heterooligomerization; cell-matrix adhesion; positive regulation of axon extension; positive regulation of transcription, DNA-dependent; negative regulation of smooth muscle cell migration; protein amino acid phosphorylation; myelin formation; positive regulation of MAP kinase activity; positive regulation of cell proliferation; fibril organization and biogenesis; protein kinase B signaling cascade; negative regulation of neuron apoptosis; cell cycle arrest; positive regulation of cell-matrix adhesion; positive regulation of BMP signaling pathway; integrin-mediated signaling pathway; cell aging; positive regulation of dendrite morphogenesis; positive regulation of myoblast differentiation; myelination in the peripheral nervous system; establishment and/or maintenance of epithelial cell polarity; cell proliferation; positive regulation of osteoblast differentiation; positive regulation of protein kinase B signaling cascade; peptidyl-serine phosphorylation; ureteric bud branching; regulation of actin cytoskeleton organization and biogenesis; negative regulation of protein kinase activity; nerve development; positive regulation of phosphorylation; neurite morphogenesis; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:
This gene encodes a protein with a kinase-like domain and four ankyrin-like repeats. The encoded protein associates at the cell membrane with the cytoplasmic domain of beta integrins, where it regulates integrin-mediated signal transduction. Activity of this protein is important in the epithelial to mesenchymal transition, and over-expression of this gene is implicated in tumor growth and metastasis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jun 2013]
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.
