Product Name:	Human PML (Protein PML) ELISA Kit
Product Code:	HUFI07150
Size:	96 Assays
Alias:	PML ELISA Kit, Protein PML ELISA Kit, Promyelocytic leukemia protein ELISA Kit, RING finger protein 71 ELISA Kit, Tripartite motif-containing protein 19 ELISA Kit, MYL ELISA Kit, PP8675 ELISA Kit, RNF71 ELISA Kit, TRIM19 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PML (Protein PML) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PML (Protein PML) and the recovery rates were calculated by comparing the measured value to the expected amount of Human PML (Protein PML) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-104	95
EDTA plasma(n=5)	90-92	91
heparin plasma(n=5)	86-105	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PML (Protein PML) 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)	88-103%	85-101%	90-102%
EDTA plasma(n=5)	86-99%	87-101%	89-99%
UFH plasma(n=5)	83-96%	80-99%	80-94%

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 Protein Function:	PML: a zinc-finger protein that can regulate transcription and can localize to nuclear bodies. Cytoplasmic forms regulate glycolysis by inhibiting the tetrameric form of PKM2. Together with SATB1, involved in local chromatin-loop remodeling and gene expression regulation at the MHC-I locus. Interacts with SIRT1, TOPBP1, TRIM27 and TRIM69. Sumoylated forms interact with SATB1 and localize to the PML nuclear bodies. Sumoylation on three sites is required for nuclear body formation. Sumoylation on Lys-160 is a prerequisite for sumoylation on Lys-65. The B1 box and the RING finger are also required for localization to nuclear bodies. May play an important role in recruitment of ELF4 into PML nuclear bodies. A chromosomal aberration involving PML can cause acute promyelocytic leukemia (APL). Seven alternatively-spliced human isoforms have been reported.
UniProt Protein Details:	Protein type:Ubiquitin conjugating system; Tumor suppressor; Transcription factor; Nucleolus; Oncoprotein Chromosomal Location of Human Ortholog: 15q22 Cellular Component: nucleoplasm; PML body; nuclear membrane; nuclear matrix; early endosome membrane; cytoplasm; nucleolus; nucleus; cytosol Molecular Function:protein binding; protein homodimerization activity; SUMO binding; DNA binding; zinc ion binding; protein heterodimerization activity; ubiquitin protein ligase binding; transcription coactivator activity; SMAD binding; cobalt ion binding Biological Process: retinoic acid receptor signaling pathway; proteasomal ubiquitin-dependent protein catabolic process; viral reproduction; apoptosis; positive regulation of apoptosis involved in mammary gland involution; regulation of MHC class I biosynthetic process; PML body organization and biogenesis; SMAD protein nuclear translocation; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; entrainment of circadian clock by photoperiod; negative regulation of cell proliferation; positive regulation of histone deacetylation; regulation of transcription, DNA-dependent; transforming growth factor beta receptor signaling pathway; response to gamma radiation; protein complex assembly; negative regulation of mitotic cell cycle; circadian regulation of gene expression; maintenance of protein localization in nucleus; cell cycle arrest; defense response to virus; negative regulation of telomere maintenance via telomerase; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; response to UV; caspase activation; regulation of protein amino acid phosphorylation; cell fate commitment; protein stabilization; transcription, DNA-dependent; cytokine and chemokine mediated signaling pathway; common-partner SMAD protein phosphorylation; regulation of circadian rhythm; negative regulation of telomerase activity; endoplasmic reticulum calcium ion homeostasis; negative regulation of angiogenesis; DNA damage response, signal transduction resulting in induction of apoptosis; response to cytokine stimulus; response to hypoxia; innate immune response; myeloid cell differentiation; negative regulation of translation in response to oxidative stress; negative regulation of cell growth; positive regulation of defense response to virus by host; negative regulation of transcription, DNA-dependent; induction of apoptosis by oxidative stress; protein targeting
NCBI Summary:	The protein encoded by this gene is a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. This phosphoprotein localizes to nuclear bodies where it functions as a transcription factor and tumor suppressor. Its expression is cell-cycle related and it regulates the p53 response to oncogenic signals. The gene is often involved in the translocation with the retinoic acid receptor alpha gene associated with acute promyelocytic leukemia (APL). Extensive alternative splicing of this gene results in several variations of the protein's central and C-terminal regions; all variants encode the same N-terminus. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:	P29590
NCBI GenInfo Identifier:	15451765
NCBI Gene ID:	5371
NCBI Accession:	NP_150242
UniProt Related Accession:	P29590
Molecular Weight:	97.516
NCBI Full Name:	protein PML isoform 9
NCBI Synonym Full Names:	PML nuclear body scaffold
NCBI Official Symbol:	PML
NCBI Official Synonym Symbols:	MYL; RNF71; PP8675; TRIM19
NCBI Protein Information:	protein PML
UniProt Protein Name:	Protein PML
UniProt Synonym Protein Names:	Promyelocytic leukemia protein; RING finger protein 71; Tripartite motif-containing protein 19
Protein Family:	Protein
UniProt Gene Name:	PML
UniProt Entry Name:	PML_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.