Human PIM1 ELISA Kit (HUFI02972)

Product Name:	Human PIM1 ELISA Kit
Product Code:	HUFI02972
Size:	96 Assays
Alias:	PIM-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PIM-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.180ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PIM-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PIM-1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-104	96
EDTA plasma(n=5)	85-103	97
UFH plasma(n=5)	86-100	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PIM-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	87-104%	89-100%	86-105%
EDTA plasma(n=5)	83-96%	83-101%	83-97%
UFH plasma(n=5)	88-98%	81-99%	87-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P11309

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.
UniProt Protein Details:	Protein type:Protein kinase, CAMK; Protein kinase, Ser/Thr (non-receptor); Oncoprotein; Kinase, protein; EC 2.7.11.1; CAMK group; PIM family Chromosomal Location of Human Ortholog: 6p21.2 Cellular Component: cytoplasm Molecular Function:ATP binding; manganese ion binding; protein binding; protein serine/threonine kinase activity; ribosomal small subunit binding; transcription factor binding Biological Process: cell proliferation; multicellular organismal development; negative regulation of apoptosis; negative regulation of transcription factor activity; protein amino acid autophosphorylation; protein amino acid phosphorylation
NCBI Summary:	The protein encoded by this gene belongs to the Ser/Thr protein kinase family, and PIM subfamily. This gene is expressed primarily in B-lymphoid and myeloid cell lines, and is overexpressed in hematopoietic malignancies and in prostate cancer. It plays a role in signal transduction in blood cells, contributing to both cell proliferation and survival, and thus provides a selective advantage in tumorigenesis. Both the human and orthologous mouse genes have been reported to encode two isoforms (with preferential cellular localization) resulting from the use of alternative in-frame translation initiation codons, the upstream non-AUG (CUG) and downstream AUG codons (PMIDs:16186805, 1825810).[provided by RefSeq, Aug 2011]
UniProt Code:	P11309
NCBI GenInfo Identifier:	83305339
NCBI Gene ID:	5292
NCBI Accession:	P11309.3
UniProt Secondary Accession:	P11309,Q38RT9, Q5T7H7, Q96RG3,
UniProt Related Accession:	P11309
Molecular Weight:	35,686 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:	PIM
NCBI Protein Information:	serine/threonine-protein kinase pim-1
UniProt Protein Name:	Serine/threonine-protein kinase pim-1
Protein Family:	Protein
UniProt Gene Name:	PIM1
UniProt Entry Name:	PIM1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.

Human PIM1 ELISA Kit (HUFI02972)

Description