Rat Integrin-linked protein kinase (Ilk) ELISA Kit
- SKU:
- RTEB1112
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q99J82
- Range:
- 0.156-10 ng/ml
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
Frequently bought together:
Description
| 商品名: | Rat Integrin-linked protein kinase (Ilk) ELISA Kit |
| 製品コード: | RTEB1112 |
| エイリアス: | Integrin-linked protein kinase, Ilk, 2.7.11.1 |
| Uniprot: | Q99J82 |
| 反応性: | Rat |
| 範囲: | 0.156-10 ng/ml |
| 検出方法: | Sandwich |
| サイズ: | 96 Assay |
| 保管所: | Please see kit components below for exact storage details |
| ノート: | 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] |
| UniProt Code: | Q99J82 |
| NCBI GenInfo Identifier: | 62420873 |
| NCBI Gene ID: | 3611 |
| NCBI Accession: | NP_001014794.1 |
| UniProt Secondary Accession: | Q99J82,O55222, Q99J82 |
| UniProt Related Accession: | Q99J82,Q13418 |
| Molecular Weight: | 51kDa |
| NCBI Full Name: | integrin-linked protein kinase isoform 1 |
| NCBI Synonym Full Names: | integrin linked kinase |
| NCBI Official Symbol: | ILK |
| NCBI Official Synonym Symbols: | P59; ILK-1; ILK-2; p59ILK; HEL-S-28 |
| NCBI Protein Information: | integrin-linked protein kinase |
| UniProt Protein Name: | Integrin-linked protein kinase |
| UniProt Synonym Protein Names: | 59 kDa serine/threonine-protein kinase; ILK-1; ILK-2; p59ILK |
| Protein Family: | Integrin-linked protein kinase |
| UniProt Gene Name: | ILK |
| UniProt Entry Name: | ILK_HUMAN |
| 成分 | 額 | 保管所 |
| 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 | - |
その他の必要な材料と設備：
- 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
*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。
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.
| ステップ | プロトコル |
| 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. |
ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。
| サンプルタイプ: | プロトコル: |
| 血清: | 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. |
| プラズマ: | 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. |
| 尿および脳脊髄液: | 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. |
| 細胞培養上清: | 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. |
| 細胞溶解物: | 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. |
| 組織ホモジネート: | 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. |
| 組織溶解物: | 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. |
| 母乳: | 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. |