Rat Cell Biology ELISA Kits 3

Rat Proto-oncogene serine/threonine-protein kinase pim-1 (Pim1) ELISA Kit

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SKU:
RTEB1313
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P26794
Range:
0.156-10 ng/mL
ELISA Type:
Sandwich
Reactivity:
Rat

Description

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Product Name:Rat Proto-oncogene serine/threonine-protein kinase pim-1 (Pim1) ELISA Kit
Product Code:RTEB1313
Alias:Proto-oncogene serine/threonine-protein kinase pim-1, Pim1, Pim-1, 2.7.11.1
Uniprot:P26794
Reactivity:Rat
Range:0.156-10 ng/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:Pim1: a proto-oncogene serine/threonine kinase involved in cell survival and cell proliferation and thus providing a selective advantage in tumorigenesis. Exerts its oncogenic activity through: the regulation of MYC transcriptional activity, the regulation of cell cycle progression and by phosphorylation and inhibition of proapoptotic proteins (BAD, MAP3K5, FOXO3). Phosphorylation of MYC leads to an increase of MYC protein stability and thereby an increase of transcriptional activity. The stabilization of MYC exerced by PIM1 might explain partly the strong synergism between these two oncogenes in tumorigenesis. Mediates survival signaling through phosphorylation of BAD, which induces release of the anti-apoptotic protein Bcl- X(L)/BCL2L1. Phosphorylation of ASK1 an other proapoptotic protein, by PIM1, significantly decreases ASK1 kinase activity and inhibits MAP3K5-mediated phosphorylation of JNK and JNK/p38MAPK subsequently reducing caspase-3 activation and cell apoptosis. Stimulates cell cycle progression at the G1-S and G2-M transitions by phosphorylation of CDC25A and CDC25C. Phosphorylation of p21Cip1, a regulator of cell cycle progression at G1, results in the relocation of p21Cip1 to the cytoplasm and enhanced p21Cip1 protein stability. Promote cell cycle progression and tumorigenesis by down-regulating expression of a regulator of cell cycle progression, p27Kip1, at both transcriptional and post- translational levels. Phosphorylation of p27Kip1,induces 14-3-3- proteins binding, nuclear export and proteasome-dependent degradation. May affect the structure or silencing of chromatin by phosphorylating HP1 gamma/CBX3. Acts also as a regulator of homing and migration of bone marrow cells involving functional interaction with the CXCL12-CXCR4 signaling axis. Isoform 2 is isolated as a monomer whereas isoform 1 complexes with other proteins. Binds to RP9. Isoform 1, but not isoform 2, binds BMX. Isoform 2 interacts with p27Kip1 and FOXO3. Interacts with BAD. Interacts with PPP2CA; this interaction promotes dephosphorylation of PIM1, ubiquitination and proteasomal degradation. Interacts with HSP90, this interaction stabilizes PIM1 protein levels. Ubiquitinated form interacts with HSP70 and promotes its proteosomal degradation. Strongly induced in leukocytes by the JAK/STAT pathway in response to cytokines. Induced by different cellular stresses, heat shock and cytotoxic agents. Expressed primarily in cells of the hematopoietic and germline lineages. 2 isoforms of the human protein are produced by alternative initiation. Both isoforms are expressed in prostate cancer cell lines.Protein type: EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, CAMK; Oncoprotein; CAMK group; PIM familyCellular Component: cytoplasm; plasma membrane; nucleusMolecular Function: protein serine/threonine kinase activity; manganese ion binding; transcription factor binding; ATP binding; ribosomal small subunit bindingBiological Process: cell proliferation; apoptosis; protein amino acid autophosphorylation; negative regulation of transcription factor activity; regulation of mitotic cell cycle; hyaluronan metabolic process; protein amino acid phosphorylation; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; negative regulation of apoptosis
UniProt Protein Details:
NCBI Summary:serine threonine protein kinase; 2.8 kb transcript is detected in somatic cells and a 2.3 kb transcript using an alternate polyadenylation signal is expressed in testis [RGD, Feb 2006]
UniProt Code:P26794
NCBI GenInfo Identifier:8393959
NCBI Gene ID:24649
NCBI Accession:NP_058730.1
UniProt Secondary Accession:P26794
UniProt Related Accession:P26794
Molecular Weight:35,631 Da
NCBI Full Name:serine/threonine-protein kinase pim-1
NCBI Synonym Full Names:Pim-1 proto-oncogene, serine/threonine kinase
NCBI Official Symbol:Pim1  
NCBI Official Synonym Symbols:
NCBI Protein Information:serine/threonine-protein kinase pim-1; pim-1 oncogene; proviral integration site 1; non-specific serine/threonine protein kinase; proto-oncogene serine/threonine-protein kinase Pim-1
UniProt Protein Name:Serine/threonine-protein kinase pim-1
UniProt Synonym Protein Names:
Protein Family:Protein
UniProt Gene Name:Pim1  
UniProt Entry Name:PIM1_RAT
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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