Mouse Lipc ELISA Kit (MOEB0585)- High Sensitivity

Product Name:	Mouse Hepatic triacylglycerol lipase (Lipc) ELISA Kit
SKU:	MOEB0585
Size:	96T
Target:	Mouse Hepatic triacylglycerol lipase (Lipc)
Synonyms:	Lipase member C, HL, Hpl
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	1.56-100ng/mL
Sensitivity:	0.83ng/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Hepatic lipase has the capacity to catalyze hydrolysis of phospholipids, mono-, di-, and triglycerides, and acyl-CoA thioesters. It is an important enzyme in HDL metabolism. Hepatic lipase binds heparin.

Uniprot:	P27656
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Hepatic triacylglycerol lipase
Research Area:	Cardiovascular
Subcellular Location:	Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	LIPC: Hepatic lipase has the capacity to catalyze hydrolysis of phospholipids, mono-, di-, and triglycerides, and acyl-CoA thioesters. It is an important enzyme in HDL metabolism. Hepatic lipase binds heparin. Defects in LIPC are the cause of hepatic lipase deficiency (HL deficiency). A disorder characterized by elevated levels of beta-migrating very low density lipoproteins, and abnormally triglyceride-rich low and high density lipoproteins. Belongs to the AB hydrolase superfamily. Lipase family.
UniProt Protein Details:	Protein type:Phospholipase; EC 3.1.1.3; Secreted; Secreted, signal peptide; Lipid Metabolism - glycerolipid Cellular Component: extracellular space; microvillus; cell surface; early endosome; late endosome; extracellular region Molecular Function:heparan sulfate proteoglycan binding; heparin binding; triacylglycerol lipase activity; protein binding; phospholipase A1 activity; hydrolase activity; low-density lipoprotein binding; lipase activity; transferase activity, transferring acyl groups; acylglycerol lipase activity; apolipoprotein binding; lipid binding; lysophospholipase activity Biological Process: cholesterol metabolic process; neutral lipid catabolic process; cholesterol transport; glycerophospholipid catabolic process; fatty acid metabolic process; protein oligomerization; phosphatidylcholine metabolic process; cholesterol homeostasis; triacylglycerol metabolic process; phosphatidic acid metabolic process; phosphatidylserine metabolic process; heparan sulfate proteoglycan biosynthetic process; triacylglycerol catabolic process; lipid metabolic process; phosphatidylethanolamine metabolic process; lipid catabolic process; fatty acid biosynthetic process
UniProt Code:	P27656
NCBI GenInfo Identifier:	126309
NCBI Gene ID:	15450
NCBI Accession:	P27656.1
UniProt Related Accession:	P27656
Molecular Weight:
NCBI Full Name:	Hepatic triacylglycerol lipase
NCBI Synonym Full Names:	lipase, hepatic
NCBI Official Symbol:	Lipc
NCBI Official Synonym Symbols:	HL; Hpl; AI256194
NCBI Protein Information:	hepatic triacylglycerol lipase
UniProt Protein Name:	Hepatic triacylglycerol lipase
UniProt Synonym Protein Names:	Lipase member C
UniProt Gene Name:	Lipc
UniProt Entry Name:	LIPC_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.