Mouse PHD finger protein 1 (Phf1) ELISA Kit

Product Name:	Mouse PHD finger protein 1 (Phf1) ELISA Kit
SKU:	MOEB1162
Size:	96T
Target:	Mouse PHD finger protein 1 (Phf1)
Synonyms:	Polycomb-like protein 1, T-complex testis-expressed 3, mPCl1, Protein PHF1, Plc1, Tctex-3, Tctex3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.312-20ng/mL
Sensitivity:	0.156ng/mL

Intra CV:

6.8%

Inter CV:

8.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	100-110%	112-122%	97-110%	90-100%
EDTA Plasma(N=5)	108-118%	80-92%	107-117%	109-118%
Heparin Plasma(N=5)	114-123%	108-118%	96-108%	100-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	89	83-95
Plasma	91	85-97

Function:

Polycomb group (PcG) that specifically binds histone H3 trimethylated at 'Lys-36' (H3K36me3) and recruits the PRC2 complex. Involved in DNA damage response and is recruited at double-strand breaks (DSBs). Acts by binding to H3K36me3, a mark for transcriptional activation, and recruiting the PRC2 complex: it is however unclear whether recruitment of the PRC2 complex to H3K36me3 leads to enhance or inhibit H3K27me3 methylation mediated by the PRC2 complex. According to some reports, PRC2 recruitment by PHF1 promotes H3K27me3 and subsequent gene silencing by inducing spreading of PRC2 and H3K27me3 into H3K36me3 loci (PubMed:18086877). According to other reports, PHF1 recruits the PRC2 complex at double-strand breaks (DSBs) and inhibits the activity of PRC2. Regulates p53/TP53 stability and prolonges its turnover: may act by specifically binding to a methylated from of p53/TP53.

Uniprot:	Q9Z1B8
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse PHD finger protein 1
Sub Unit:	Associated component of the PRC2 complex. Interacts with p53/TP53 (By similarity). Interacts with CHMP1.
Subcellular Location:	Nucleus Cytoplasm Cytoskeleton Microtubule organizing center Centrosome Localizes specifically to the promoters of numerous target genes. Localizes to double-strand breaks (DSBs) sites following DNA damage. Colocalizes with NEK6 in the centrosome (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PHF1: Transcriptional repressor. May promote methylation of histone H3 on 'Lys-27' by the PRC2/EED-EZH2 complex. 2 isoforms of the human protein are produced by alternative splicing.Protein type: Transcription factorCellular Component: nucleoplasm; cytoskeleton; ESC/E(Z) complex; cytoplasm; nucleusMolecular Function: protein binding; zinc ion binding; metal ion binding; chromatin binding; methylated histone residue bindingBiological Process: regulation of transcription, DNA-dependent; transcription, DNA-dependent; chromatin modification; response to DNA damage stimulus
UniProt Protein Details:
NCBI Summary:	The protein encoded by this gene belongs to the polycomb-like protein family, which is a component of polycomb repressive complex-2. This complex represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 and is required for the regulation of developmental genes including homeotic genes. The gene is expressed primarily in testis tissue. Small interfering RNA-mediated knockdown in cultured cell lines results in changes in homeotic gene expression coincident with alterations in promoter methylation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Oct 2014]
UniProt Code:	Q9Z1B8
NCBI GenInfo Identifier:	111185928
NCBI Gene ID:	21652
NCBI Accession:	NP_033369.2
UniProt Secondary Accession:	Q9Z1B8,O54808
UniProt Related Accession:	Q9Z1B8
Molecular Weight:	61,140 Da
NCBI Full Name:	PHD finger protein 1
NCBI Synonym Full Names:	PHD finger protein 1
NCBI Official Symbol:	Phf1
NCBI Official Synonym Symbols:	PHF2; mPcl1; Tctex3; Tctex-3; AW557215; D17Ertd455e
NCBI Protein Information:	PHD finger protein 1; polycomb-like protein 1; t-complex testis-expressed 3
UniProt Protein Name:	PHD finger protein 1
UniProt Synonym Protein Names:	Polycomb-like protein 1; mPCl1; T-complex testis-expressed 3
Protein Family:	PHD finger protein
UniProt Gene Name:	Phf1
UniProt Entry Name:	PHF1_MOUSE

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.