Rat Serine/threonine-protein kinase pim-3 (Pim3) ELISA Kit
The Rat Serine/Threonine Protein Kinase Pim-3 (PIM3) ELISA Kit is specially designed for the precise quantitative measurement of PIM3 levels in rat serum, plasma, and tissue lysates. This kit offers excellent sensitivity and specificity, ensuring accurate and reproducible results for various research applications.PIM3 is a key protein kinase that plays a crucial role in cell proliferation, apoptosis, and oncogenic transformation. It is involved in several signaling pathways and is often dysregulated in cancer, making it a potential therapeutic target for cancer treatment.
By utilizing the Rat PIM3 ELISA Kit, researchers can effectively study the role of PIM3 in disease pathogenesis, identify potential biomarkers for cancer diagnosis, and develop targeted therapies for cancer treatment. Get reliable and insightful data with this highly reliable ELISA kit from Assay Genie.
Product Name:
Rat Serine/threonine-protein kinase pim-3 (Pim3) ELISA Kit
SKU:
RTEB1533
Size:
96T
Target:
Rat Serine/threonine-protein kinase pim-3 (Pim3)
Synonyms:
Kinase induced by depolarization, Protein kinase Kid-1, Kid1
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.312-20ng/mL
Sensitivity:
0.169ng/mL
Intra CV:
4.9%
Inter CV:
6.2%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
104-112%
107-117%
91-101%
97-106%
EDTA Plasma(N=5)
104-114%
89-99%
91-104%
105-115%
Heparin Plasma(N=5)
109-119%
81-90%
85-94%
93-102%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
94
88-100
Plasma
96
90-102
Function:
Proto-oncogene with serine/threonine kinase activity that can prevent apoptosis and promote cell survival and protein translation. May contribute to tumorigenesis through: the delivery of survival signaling through phosphorylation of BAD which induces release of the anti-apoptotic protein Bcl-X(L), the regulation of cell cycle progression and protein synthesis and by regulation of MYC transcriptional activity. Additionally to this role on tumorigenesis, can also negatively regulate insulin secretion by inhibiting the activation of MAPK1/3 (ERK1/2), through SOCS6. Involved also in the control of energy metabolism and regulation of AMPK activity in modulating MYC and PPARGC1A protein levels and cell growth.
Uniprot:
O70444
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Serine/threonine-protein kinase pim-3
Sub Unit:
Interacts with BAD. Interacts with PPP2CA; this interaction promotes dephosphorylation of PIM3, ubiquitination and proteasomal degradation. Interacts with SOCS6.
Subcellular Location:
Cytoplasm
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Pim3: a Ca(2 )/calmodulin-dependent protein kinase of the PIM family. Highly expressed in hematopoietic tissues, in leukemia and lymphoma cell lines, testis, small intestine, colon and colorectal adenocarcinoma cells.Protein type: Protein kinase, CAMK; Kinase, protein; EC 2.7.11.1; Oncoprotein; Protein kinase, Ser/Thr (non-receptor); CAMK group; PIM familyCellular Component: cytoplasmMolecular Function: protein serine/threonine kinase activity; ATP bindingBiological Process: apoptosis; histone phosphorylation; protein amino acid autophosphorylation; regulation of mitotic cell cycle; protein amino acid phosphorylation; negative regulation of apoptosis
UniProt Protein Details:
NCBI Summary:
catalyzes both autophosphorylation and phosphorylation of other proteins; may play a role in synaptic plasticity [RGD, Feb 2006]
serine/threonine-protein kinase pim-3; pim-3 oncogene; protein kinase Kid-1; proviral integration site 3; serine threonine kinase pim3; kinase induced by depolarization; serine/threonine protein kinase pim-3
UniProt Protein Name:
Serine/threonine-protein kinase pim-3
UniProt Synonym Protein Names:
Kinase induced by depolarization; Protein kinase Kid-1
Protein Family:
Serine/threonine-protein kinase
UniProt Gene Name:
Pim3
UniProt Entry Name:
PIM3_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.
