Product Name:	Human Hepcidin (HAMP) ELISA Kit
SKU:	HUEB2077
Size:	96T
Target:	Human Hepcidin (HAMP)
Synonyms:	Liver-expressed antimicrobial peptide 1, Putative liver tumor regressor, LEAP-1, PLTR, UNQ487/PRO1003, HEPC, LEAP1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.18-12ng/mL
Sensitivity:	0.094ng/mL

Intra CV:

6.9%

Inter CV:

8.9%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	89-99%	86-98%	101-110%	91-99%
EDTA Plasma(N=5)	101-112%	83-93%	88-98%	86-99%
Heparin Plasma(N=5)	102-112%	91-101%	106-119%	108-117%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	91	85-97
Plasma	93	87-99

Function:

Has strong antimicrobial activity against E.coli ML35P N.cinerea and weaker against S.epidermidis, S.aureus and group b streptococcus bacteria. Active against the fungus C.albicans. No activity against P.aeruginosa (PubMed:11113131, PubMed:11034317).

Uniprot:	P81172
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Hepcidin
Subcellular Location:	Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	HAMP: Seems to act as a signaling molecule involved in the maintenance of iron homeostasis. Seems to be required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages. Defects in HAMP are the cause of hemochromatosis type 2B (HFE2B); also known as juvenile hemochromatosis (JH). HFE2B is a disorder of iron metabolism with excess deposition of iron in the tissues, bronze skin pigmentation, hepatic cirrhosis, arthropathy and diabetes. The most common symptoms of hemochromatosis type 2 at presentation are hypogonadism and cardiomyopathy. Belongs to the hepcidin family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 19q13.1 Cellular Component: apical cortex; extracellular region Molecular Function:hormone activity Biological Process: killing of cells of another organism; cellular iron ion homeostasis; defense response to bacterium; immune response; defense response to fungus Disease: Hemochromatosis, Type 2b
NCBI Summary:	The product encoded by this gene is involved in the maintenance of iron homeostasis, and it is necessary for the regulation of iron storage in macrophages, and for intestinal iron absorption. The preproprotein is post-translationally cleaved into mature peptides of 20, 22 and 25 amino acids, and these active peptides are rich in cysteines, which form intramolecular bonds that stabilize their beta-sheet structures. These peptides exhibit antimicrobial activity against bacteria and fungi. Mutations in this gene cause hemochromatosis type 2B, also known as juvenile hemochromatosis, a disease caused by severe iron overload that results in cardiomyopathy, cirrhosis, and endocrine failure. [provided by RefSeq, Oct 2014]
UniProt Code:	P81172
NCBI GenInfo Identifier:	10720397
NCBI Gene ID:	57817
NCBI Accession:	P81172.2
UniProt Related Accession:	P81172
Molecular Weight:
NCBI Full Name:	Hepcidin
NCBI Synonym Full Names:	hepcidin antimicrobial peptide
NCBI Official Symbol:	HAMP
NCBI Official Synonym Symbols:	HEPC; PLTR; HFE2B; LEAP1
NCBI Protein Information:	hepcidin
UniProt Protein Name:	Hepcidin
UniProt Synonym Protein Names:	Liver-expressed antimicrobial peptide 1; LEAP-1; Putative liver tumor regressor
Protein Family:	Hepcidin
UniProt Gene Name:	HAMP
UniProt Entry Name:	HEPC_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.