Product Name:	Human Mucin-1 (MUC1) ELISA Kit
Product Code:	HUEB0647
Alias:	Mucin-1, MUC-1, Breast carcinoma-associated antigen DF3, Carcinoma-associated mucin, Episialin, H23AG, PEMT, Peanut-reactive urinary mucin, PUM, Polymorphic epithelial mucin, PEM, Tumor-associated epithelial membrane antigen, EMA, Tumor-associated mucin, MUC1, PUM, CA 15-3, PEM, Tumor-associated epithelial membrane antigen, EMA, Tumor-associated mucin, CD227
Uniprot:	P15941
Reactivity:	Human
Range:	0.156-10 ng/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	MUC1: a large cell surface glycoprotein expressed by most glandular and ductal epithelial cells and some hematopoietic cell lineages. Plays a role in adhesion and cell-cell interactions, metastasis and signaling. May provide a protective layer on epithelial surfaces. Direct or indirect interaction with actin cytoskeleton. Its cytoplasmic tail (MUC1CT) is involved in several signaling pathways, including those involving Ras, beta-catenin, p120 catenin, p53 and estrogen receptor alpha. MUC1CT also forms complexes with transcription factors, and then translocates to the nucleus by an unknown mechanism, where it is believed to influence the transcription of their target genes. MUC1CT has also been proposed to localize to mitochondrial membranes under conditions of genotoxic stress, where it attenuates the apoptotic pathway in response and confers resistance to apoptosis-inducing drugs. Aberrantly glycosylated forms expressed in human epithelial tumors, such as breast or ovarian cancer and also in non-epithelial tumor cells. Nine alternatively spliced isoforms have been described. Isoforms 5 and 9 are secreted. Isoform 7, expressed only in tumor cells, is a receptor and binds the secreted isoform 5. The binding induces the phosphorylation of the isoform 7, alters cellular morphology and initiates cell signaling. Can bind to GRB2 adapter protein.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Motility/polarity/chemotaxis; Actin-binding; Membrane protein, integral; Tumor suppressor; Cell adhesion Chromosomal Location of Human Ortholog: 1q21 Cellular Component: extracellular space; cell surface; integral to plasma membrane; Golgi lumen; cytoplasm; nuclear chromatin; apical plasma membrane; vesicle Molecular Function:protein binding; p53 binding; transcription cofactor activity Biological Process: protein amino acid O-linked glycosylation; DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator; cellular protein metabolic process; regulation of transcription from RNA polymerase II promoter in response to stress; O-glycan processing; post-translational protein modification; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest Disease: Medullary Cystic Kidney Disease 1
NCBI Summary:	This gene encodes a membrane-bound protein that is a member of the mucin family. Mucins are O-glycosylated proteins that play an essential role in forming protective mucous barriers on epithelial surfaces. These proteins also play a role in intracellular signaling. This protein is expressed on the apical surface of epithelial cells that line the mucosal surfaces of many different tissues including lung, breast stomach and pancreas. This protein is proteolytically cleaved into alpha and beta subunits that form a heterodimeric complex. The N-terminal alpha subunit functions in cell-adhesion and the C-terminal beta subunit is involved in cell signaling. Overexpression, aberrant intracellular localization, and changes in glycosylation of this protein have been associated with carcinomas. This gene is known to contain a highly polymorphic variable number tandem repeats (VNTR) domain. Alternate splicing results in multiple transcript variants.[provided by RefSeq, Feb 2011]
UniProt Code:	P15941
NCBI GenInfo Identifier:	296439295
NCBI Gene ID:	4582
NCBI Accession:	P15941.3
UniProt Secondary Accession:	P15941,P13931, P15942, P17626, A5YRV1, A6ZID9, A6ZIE0 B1AVQ8, B1AVR0, B6ECA1, E7ESE5, E7EUG9,
UniProt Related Accession:	P15941
Molecular Weight:
NCBI Full Name:	Mucin-1
NCBI Synonym Full Names:	mucin 1, cell surface associated
NCBI Official Symbol:	MUC1
NCBI Official Synonym Symbols:	EMA; MCD; PEM; PUM; KL-6; MAM6; MCKD; PEMT; CD227; H23AG; MCKD1; MUC-1; ADMCKD; ADMCKD1; CA 15-3; MUC-1/X; MUC1/ZD; MUC-1/SEC
NCBI Protein Information:	mucin-1; episialin; DF3 antigen; H23 antigen; cancer antigen 15-3; krebs von den Lungen-6; mucin 1, transmembrane; carcinoma-associated mucin; polymorphic epithelial mucin; peanut-reactive urinary mucin; tumor associated epithelial mucin; breast carcinoma-associated antigen DF3; tumor-associated epithelial membrane antigen; Medullary cystic kidney disease, autosomal dominant
UniProt Protein Name:	Mucin-1
UniProt Synonym Protein Names:	Breast carcinoma-associated antigen DF3; Cancer antigen 15-3; CA 15-3; Carcinoma-associated mucin; Episialin; H23AG; Krebs von den Lungen-6; KL-6; PEMT; Peanut-reactive urinary mucin; PUM; Polymorphic epithelial mucin; PEM; Tumor-associated epithelial membrane antigen; EMA; Tumor-associated mucin; CD_antigen: CD227Cleaved into the following 2 chains:Mucin-1 subunit alpha; MUC1-NT; MUC1-alpha; Mucin-1 subunit beta; MUC1-betaAlternative name(s):MUC1-CT
Protein Family:	Mucin
UniProt Gene Name:	MUC1
UniProt Entry Name:	MUC1_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 Mucin-1 / MUC1 ELISA Kit	Anti-MUC1 Antibody (CAB0333)
Human MUC1 (Mucin 1) CLIA Kit (HUES00408)	Anti-MUC1 Antibody (CAB19081)
Human MUC1 (Mucin 1) ELISA Kit (HUES01780)	MUC1 Antibody (PACO01119)
	Phospho-MUC1 (Y1229) Antibody (PACO04364)
	Phospho-MUC1 (Y1243) Antibody (PACO04365)