Rat Phospholipase D1 (Pld1) ELISA Kit

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SKU:
RTEB0938
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P70496
ELISA Type:
Sandwich
Reactivity:
Rat
€0.00
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Description

system_update_altデータシートsystem_update_alt安全性データシート
商品名:Rat Phospholipase D1 (Pld1) ELISA Kit
製品コード:RTEB0938
エイリアス:Phospholipase D1, PLD 1, rPLD1, Choline phosphatase 1, Phosphatidylcholine-hydrolyzing phospholipase D1, Pld1, 3.1.4.4
Uniprot:P70496
反応性:Rat
範囲:Please contact us for more information
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:PLD1: a phosphatidylcholine-hydrolyzing phospholipase. Activated by ADP-ribosylated Rho and protein kinase C. Implicated as a critical step in numerous cellular pathways including membrane trafficking and the regulation polymorphonuclear leukocyte degranulation and oxidant production. May be involved in the regulation of perinuclear intravesicular membrane traffic. Four splice-variant isoforms have been described.Protein type: Phospholipase; EC 3.1.4.4; Lipid Metabolism - ether lipid; Lipid Metabolism - glycerophospholipid; Motility/polarity/chemotaxisCellular Component: Golgi apparatus; endoplasmic reticulum membrane; Golgi cisterna; intracellular membrane-bound organelle; lysosomal membrane; late endosome membrane; Golgi membrane; membrane; lamellipodium; perinuclear region of cytoplasm; endocytic vesicle; apical plasma membrane; vesicle; endosomeMolecular Function: protein binding; phospholipase D activity; catalytic activity; phosphoinositide bindingBiological Process: response to peptide hormone stimulus; defense response to Gram-positive bacterium; phospholipid catabolic process; metabolic process; phosphatidic acid biosynthetic process; regulation of microvillus biogenesis; phospholipid biosynthetic process; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:ubiquitous enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline [RGD, Feb 2006]
UniProt Code:P70496
NCBI GenInfo Identifier:25742819
NCBI Gene ID:25096
NCBI Accession:
UniProt Secondary Accession:P70496
UniProt Related Accession:P70496
Molecular Weight:
NCBI Full Name:phospholipase D1
NCBI Synonym Full Names:phospholipase D1
NCBI Official Symbol:Pld1  
NCBI Official Synonym Symbols:Plda; Pldb; Pld1a; Pld1b  
NCBI Protein Information:phospholipase D1
UniProt Protein Name:Phospholipase D1
UniProt Synonym Protein Names:Choline phosphatase 1; Phosphatidylcholine-hydrolyzing phospholipase D1
Protein Family:Phospholipase
UniProt Gene Name:Pld1  
UniProt Entry Name:PLD1_RAT
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.
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