Human PROC ELISA Kit (HUEB0832)- High Sensitivity

Product Name:	Human Vitamin K-dependent protein C (PROC) ELISA Kit
SKU:	HUEB0832
Size:	96T
Target:	Human Vitamin K-dependent protein C (PROC)
Synonyms:	Anticoagulant protein C, Autoprothrombin IIA, Blood coagulation factor XIV
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.195ng/mL

Intra CV:

7.5%

Inter CV:

10.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	80-93%	91-101%	95-104%	96-104%
EDTA Plasma(N=5)	87-99%	96-106%	109-117%	85-95%
Heparin Plasma(N=5)	102-112%	102-113%	92-102%	99-109%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	98	92-104
Plasma	100	94-106

Function:

Protein C is a vitamin K-dependent serine protease that regulates blood coagulation by inactivating factors Va and VIIIa in the presence of calcium ions and phospholipids (PubMed:25618265). Exerts a protective effect on the endothelial cell barrier function (PubMed:25651845).

Uniprot:	P04070
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Vitamin K-dependent protein C
Sub Unit:	Synthesized as a single chain precursor, which is cleaved into a light chain and a heavy chain held together by a disulfide bond. The enzyme is then activated by thrombin, which cleaves a tetradecapeptide from the amino end of the heavy chain; this reaction, which occurs at the surface of endothelial cells, is strongly promoted by thrombomodulin.
Research Area:	Cancer
Subcellular Location:	Secreted Golgi apparatus Endoplasmic reticulum
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PROC: Protein C is a vitamin K-dependent serine protease that regulates blood coagulation by inactivating factors Va and VIIIa in the presence of calcium ions and phospholipids. Defects in PROC are the cause of thrombophilia due to protein C deficiency, autosomal dominant (THPH3). A hemostatic disorder characterized by impaired regulation of blood coagulation and a tendency to recurrent venous thrombosis. However, many adults with heterozygous disease may be asymptomatic. Individuals with decreased amounts of protein C are classically referred to as having type I protein C deficiency and those with normal amounts of a functionally defective protein as having type II deficiency. Defects in PROC are the cause of thrombophilia due to protein C deficiency, autosomal recessive (THPH4). A hemostatic disorder characterized by impaired regulation of blood coagulation and a tendency to recurrent venous thrombosis. It results in a thrombotic condition that can manifest as a severe neonatal disorder or as a milder disorder with late-onset thrombophilia. The severe form leads to neonatal death through massive neonatal venous thrombosis. Often associated with ecchymotic skin lesions which can turn necrotic called purpura fulminans, this disorder is very rare. Belongs to the peptidase S1 family.
UniProt Protein Details:	Protein type:Protease; Apoptosis; EC 3.4.21.69 Chromosomal Location of Human Ortholog: 2q13-q14 Cellular Component: endoplasmic reticulum lumen; Golgi lumen; extracellular region Molecular Function:protein binding; serine-type endopeptidase activity; calcium ion binding Biological Process: cellular protein metabolic process; negative regulation of blood coagulation; blood coagulation; proteolysis; post-translational protein modification; peptidyl-glutamic acid carboxylation; leukocyte migration; negative regulation of apoptosis Disease: Thrombophilia Due To Protein C Deficiency, Autosomal Recessive; Thrombophilia Due To Protein C Deficiency, Autosomal Dominant
NCBI Summary:	This gene encodes a vitamin K-dependent plasma glycoprotein. The encoded protein is cleaved to its activated form by the thrombin-thrombomodulin complex. This activated form contains a serine protease domain and functions in degradation of the activated forms of coagulation factors V and VIII. Mutations in this gene have been associated with thrombophilia due to protein C deficiency, neonatal purpura fulminans, and recurrent venous thrombosis.[provided by RefSeq, Dec 2009]
UniProt Code:	P04070
NCBI GenInfo Identifier:	131067
NCBI Gene ID:	5624
NCBI Accession:	P04070.1
UniProt Secondary Accession:	P04070,Q15189, Q15190, Q16001, Q53S74, Q9UC55, B4DPQ7
UniProt Related Accession:	P04070
Molecular Weight:	461
NCBI Full Name:	Vitamin K-dependent protein C
NCBI Synonym Full Names:	protein C (inactivator of coagulation factors Va and VIIIa)
NCBI Official Symbol:	PROC
NCBI Official Synonym Symbols:	PC; APC; PROC1; THPH3; THPH4
NCBI Protein Information:	vitamin K-dependent protein C; prepro-protein C; autoprothrombin IIA; anticoagulant protein C; blood coagulation factor XIV
UniProt Protein Name:	Vitamin K-dependent protein C
UniProt Synonym Protein Names:	Anticoagulant protein C; Autoprothrombin IIA; Blood coagulation factor XIVCleaved into the following 3 chains:Vitamin K-dependent protein C light chain; Vitamin K-dependent protein C heavy chain; Activation peptide
Protein Family:	Vitamin K-dependent protein
UniProt Gene Name:	PROC
UniProt Entry Name:	PROC_HUMAN

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.

ELISA	Antibodies
Human PROC (Protein C) ELISA Kit	Anti-PROC Antibody (CAB1432)
Human PROC (Protein C) CLIA Kit (HUES00675)	Anti-Protein C Antibody (CAB4532)
Human PROC (Protein C) ELISA Kit (HUES02247)	PROC Antibody (PACO01933)
	LIPC Antibody (PACO21133)
	PRL Antibody, Biotin conjugated (PACO49905)