Human DPP1/CTSC ELISA Kit (HUFI01906)

Product Name:	Human DPP1/CTSC ELISA Kit
Product Code:	HUFI01906
Size:	96 Assays
Alias:	CTSC, DPPI, PALS, PLS, cathepsin C, EC 3.4.14.1, Cathepsin J, CPPIHMS, dipeptidyl peptidase 1, Dipeptidyl peptidase I, Dipeptidyl transferase, dipeptidyl-peptidase I, DPP1, DPPI, DPP-I, JP, JPD
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CTSC concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/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 CTSC and the recovery rates were calculated by comparing the measured value to the expected amount of Human CTSC in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-100	95
EDTA plasma(n=5)	92-103	96
UFH plasma(n=5)	88-102	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CTSC 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)	85-102%	91-99%	86-103%
EDTA plasma(n=5)	85-101%	88-96%	83-100%
UFH plasma(n=5)	83-95%	83-96%	81-100%

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

P53634

UniProt Protein Function:	CTSC: Thiol protease. Has dipeptidylpeptidase activity. Active against a broad range of dipeptide substrates composed of both polar and hydrophobic amino acids. Proline cannot occupy the P1 position and arginine cannot occupy the P2 position of the substrate. Can act as both an exopeptidase and endopeptidase. Activates serine proteases such as elastase, cathepsin G and granzymes A and B. Can also activate neuraminidase and factor XIII. Defects in CTSC are a cause of Papillon-Lefevre syndrome (PLS); also known as keratosis palmoplantaris with periodontopathia. PLS is an autosomal recessive disorder characterized by palmoplantar keratosis and severe periodontitis affecting deciduous and permanent dentitions and resulting in premature tooth loss. The palmoplantar keratotic phenotype vary from mild psoriasiform scaly skin to overt hyperkeratosis. Keratosis also affects other sites such as elbows and knees. Defects in CTSC are a cause of Haim-Munk syndrome (HMS); also known as keratosis palmoplantaris with periodontopathia and onychogryposis or Cochin Jewish disorder. HMS is an autosomal recessive disorder characterized by palmoplantar keratosis, onychogryphosis and periodontitis. Additional features are pes planus, arachnodactyly, and acroosteolysis. Defects in CTSC are a cause of aggressive periodontititis type 1 (AP1); also known as juvenile periodontitis (JPD) and prepubertal periodontitis (PPP). AP1 is characterized by severe and protracted gingival infections, leading to tooth loss. AP1 inheritance is autosomal dominant. Belongs to the peptidase C1 family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3.4.14.1; Endoplasmic reticulum; Protease Chromosomal Location of Human Ortholog: 11q14.2 Cellular Component: Golgi apparatus; extracellular space; membrane; lysosome; endoplasmic reticulum Molecular Function:identical protein binding; protein binding; protein self-association; chaperone binding; serine-type endopeptidase activity; apoptotic protease activator activity; chloride ion binding; cysteine-type peptidase activity; phosphatase binding Biological Process: response to organic substance; apoptosis; immune response; proteolysis; T cell mediated cytotoxicity; aging Disease: Periodontitis, Aggressive, 1; Papillon-lefevre Syndrome; Haim-munk Syndrome
NCBI Summary:	The protein encoded by this gene, a member of the peptidase C1 family, is a lysosomal cysteine proteinase that appears to be a central coordinator for activation of many serine proteinases in immune/inflammatory cells. It is composed of a dimer of disulfide-linked heavy and light chains, both produced from a single protein precursor, and a residual portion of the propeptide acts as an intramolecular chaperone for the folding and stabilization of the mature enzyme. This enzyme requires chloride ions for activity and can degrade glucagon. Defects in the encoded protein have been shown to be a cause of Papillon-Lefevre syndrome, an autosomal recessive disorder characterized by palmoplantar keratosis and periodontitis. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P53634
NCBI GenInfo Identifier:	88683003
NCBI Gene ID:	1075
NCBI Accession:	AAI13851.1
UniProt Secondary Accession:	P53634,Q2HIY8, Q53G93, Q71E75, Q71E76, Q7M4N9, Q7Z3G7 Q7Z5U7, Q8WY99, Q8WYA7, A8K7V2, B5MDD5,
UniProt Related Accession:	P53634
Molecular Weight:	51,854 Da
NCBI Full Name:	CTSC protein
NCBI Synonym Full Names:	cathepsin C
NCBI Official Symbol:	CTSC
NCBI Official Synonym Symbols:	JP; HMS; JPD; PLS; CPPI; DPP1; DPPI; PALS; DPP-I; PDON1
NCBI Protein Information:	dipeptidyl peptidase 1; cathepsin J; dipeptidyl peptidase I; dipeptidyl transferase; dipeptidyl-peptidase I
UniProt Protein Name:	Dipeptidyl peptidase 1
UniProt Synonym Protein Names:	Cathepsin C; Cathepsin J; Dipeptidyl peptidase I; DPP-I; DPPI; Dipeptidyl transferaseCleaved into the following 3 chains:Dipeptidyl peptidase 1 exclusion domain chain; Alternative name(s):; Dipeptidyl peptidase I exclusion domain chainDipeptidyl peptidase 1 heavy chain; Alternative name(s):; Dipeptidyl peptidase I heavy chainDipeptidyl peptidase 1 light chain; Alternative name(s):; Dipeptidyl peptidase I light chain
Protein Family:	Cathepsin
UniProt Gene Name:	CTSC
UniProt Entry Name:	CATC_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 Âµl 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 Âµl 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 DPP1/CTSC ELISA Kit (HUFI01906)

Description