Cell lysosomal lipase (acid lipase) activity colorimetric quantitative detection kit instructions

Cell lysosomal lipase (acid lipase) activity colorimetric quantitative detection kit product manual (Chinese version)

The main purpose

Cell lysosomal lipase (acid lipase) activity colorimetric quantitative detection reagent is a system designed to pass the lysosomal lipase reaction system in the presence or absence of a selective inhibitor, the substrate cholesterol oleic acid The free fatty acid after ester hydrolysis, the blue-green product after reaction with copper acetate, exhibits a change in the peak of absorption, that is, the authoritative and classical technical method for determining the enzyme activity in the cell lysis extraction sample by colorimetric method. The technology has been carefully developed and successfully tested. It is suitable for the detection of lysosomal lipase activity in various cell lysis samples (animal, human, insect, etc.). The product is strictly sterile, ready to use, simple in operation and stable in performance.

technical background

Lysosomal acid lipase (LAL; EC 3.1.1.13), also known as acid cholesteryl ester hydrolase, is one of the hydrolase in lipoprotein metabolism, containing A 372 Amino Acid glycoprotein is present in lysosomes in all tissue cells except red blood cells. In an acidic environment, by hydrolyzing cholesterol esters and triacylglycerols, it degrades intracellular lipoproteins, releases free fatty acids, and regulates lipid metabolism in cells, especially cholesterol synthesis and glycerol. Ester metabolism. Defects in lysosomal lipase will result in Wolman disease and Cholesteryl Ester Storage Disease. Lysosomal lipase has the potential to treat atherosclerosis and peripheral vascular disease. Based on the substrate cholesterol oleate (cholesterol oleate), in the presence or absence of lysosomal acid lipase specific inhibitor chlorpromazine, free fatty acid is produced by hydrolysis of lysosomal acid lipase Oleic acid (oleate), which is then reacted with the chromogenic reagent copper acetate to produce a blue-green copper salt complex. The absorbance value (715 nm wavelength) is measured spectrophotometrically to quantify the activity of the lysosomal acid lipase. . The reaction system is:

Lysosomal acid lipase

Cholesterol oleate + H2O → cholesterol + oleate

Chlorpromazine(+/—)

Oleate + cupric acetate/pyridine → cupric salt

product content

Cleaning solution (Reagent A) 120 ml

Lysate (Reagent B) 20 ml

Buffer (Reagent C) 50 ml

Substrate solution (Reagent D) 5 ml

Diluent (Reagent E) 50 ml

Reagent F 10 ml

Obligate liquid (Reagent G) 1 ml

Standard solution (Reagent H) 1 ml

Product manual 1 copy

storage method

Stored in a 4 ° C refrigerator, the diluent (Reagent E) and standard solution (Reagent H) are volatile and toxic, pay attention to the closed cap and safe operation; effective guarantee for March

User-supplied

1.5 ml centrifuge tube: container for sample handling

2 ml centrifuge tube: container for sample handling

15 ml conical centrifuge tube: container for sample preparation

Cell scraping rod: for detachment from adherent cells

DOUNCE homogenizer: for lysing cells

(Micro) Benchtop Centrifuge: for sample handling

Cuvette: a container for colorimetry

Spectrophotometer: for colorimetric analysis

Experimental procedure

• Sample preparation
• Prepare the cultured cells (1 to 5 X 10 ⁶ cells) in a 25 cm ² cell culture flask or 60 mm cell culture dish.
• Carefully add 3 ml of cleaning solution ( Reagent A ) to cover the growth surface.
• Carefully remove the cleaning solution
• Gently scrape off cells with a cell scraping rod (Note: trypsinization can be used )
• Add 3 ml of cleaning solution ( Reagent A ) and mix the cells.
• Transfer to a pre-cooled 15 ml conical centrifuge tube (note: suspension cells start from this step )
• Place in a 4°C benchtop centrifuge for 5 minutes at a speed of 300g
• Carefully remove the supernatant
• Add 1 ml of lysate ( Reagent B ) and mix thoroughly
• Vortex for 5 seconds to fully mix the cell population
• Immediately placed in the pre-cooled DOUNCE homogenizer
• Homogenize the cells with a homogenized rod in an ice bath (about 20 to 40)
• Transfer all cell homogenates into a 1.5 ml centrifuge tube
• Centrifuge in a 4°C micro tabletop centrifuge for 10 minutes at 800g
• Carefully remove the supernatant to another new pre-cooled 1.5 ml centrifuge tube
• Centrifuge in a 4°C microcentrifuge for 15 minutes at a speed of 10,000g
• Carefully remove the supernatant and retain the pellet
• Add 500 μl of buffer ( Reagent C )
• Strong vortex oscillation for 15 seconds
• Pipette 10 [mu] l for protein quantification (Note: We recommend using the Bradford Protein Assay Kit -30030.1 concentration)
• Immediately put in -70 ° C to save or place in the ice trough to continue the subsequent operations

• Preparation for measurement
• Prepare the sample to be tested (eg cell lysis sample, etc.) and place it in the ice trough
• Set the spectrophotometer (temperature 37 °C): 715nm wavelength, use the diluent (Reagent E) to zero
• Buffer ( Reagent C ) Equilibrium temperature at room temperature

Third, standard sample preparation

• Prepare 4 1.5 ml centrifuge tubes labeled 1 to 4
• Add 200 μl of diluent (Reagent E ) to tubes 2 to 4
• Pipette 400 μl of standard solution (Reagent H ) into tube 1
• Carefully transfer 200 μl of No. 1 tube standard solution (Reagent H ) to tube 2 and mix.
• Carefully remove 200 μl of No. 2 tube diluted standard solution (Reagent H ) into tube 3 and mix.
• Put the 1 to 4 tubes into the ice tank for use, avoiding the light; the standard tube concentration is shown in the table below.

• Standard curve determination

• Pipette 900 μl of diluent (Reagent E ) into a 2 ml centrifuge tube
• Add 100 μl of the above prepared standard solution (Reagent H )
• Add 200 μl of color developing solution (Reagent F )
• Vortex oscillation for 60 seconds
• Place in a mini tabletop centrifuge for 5 minutes at a speed of 1000g
• Carefully remove 1 ml of blue-green upper liquid phase into a new cuvette
• Immediately put into the spectrophotometer to detect: obtain absorbance readings
• Repeat the experiment steps 1 to 7 four times
• Construct a standard curve: the ordinate (Y-axis) is the absorbance reading; the abscissa (X-axis) is the standard oleic acid concentration (mmol/L)

• Background control

• Pipette 800 μl of buffer ( Reagent C ) into a 2 ml centrifuge tube
• Add 100 μl of substrate solution ( Reagent D )
• Add 100 μl of Diluent ( Reagent E )
• Vortex oscillation for 60 seconds
• Incubate for 30 minutes at 37 ° C
• Pipette 100 μl into a new 2 ml centrifuge tube
• Add 1 ml of diluent (Reagent E )
• Add 200 μl of color developing solution (Reagent F )
• Vortex oscillation for 60 seconds
• Place in a mini tabletop centrifuge for 5 minutes at a speed of 1000g
• Carefully remove 1 ml of the upper liquid phase into a new cuvette
• Immediately put into the spectrophotometer to detect: obtain background absorbance readings

Sixth, the total activity of the sample

• Pipette 800 μl of buffer ( Reagent C ) into a 2 ml centrifuge tube
• Add 100 μl of substrate solution ( Reagent D )
• Add 100 μl of the sample to be tested prepared above (50 μg sample protein)
• Vortex oscillation for 60 seconds
• Incubate for 30 minutes at 37 ° C
• Pipette 100 μl into a new 2 ml centrifuge tube
• Add 1 ml of diluent (Reagent E )
• Add 200 μl of color developing solution (Reagent F )
• Vortex oscillation for 60 seconds
• Place in a mini tabletop centrifuge for 5 minutes at a speed of 1000g
• Carefully remove 1 ml of blue-green upper liquid phase into a new cuvette
• Immediately put into the spectrophotometer for detection: obtain sample absorbance readings
• Actual absorbance reading: sample absorbance reading - background absorbance reading
• The sample corresponds to the oleic acid concentration (mmol/L) according to the standard curve.

• Sample non-specific activity assay

• Pipette 750 μl of buffer ( Reagent C ) into a 2 ml centrifuge tube
• Add 100 μl of substrate solution ( Reagent D )
• Add 50 μl of specific liquid (Reagent G)
• Add 100 μl of the sample to be tested prepared above (50 μg sample protein)
• Vortex oscillation for 60 seconds
• Incubate for 30 minutes at 37 ° C
• Pipette 100 μl into a new 2 ml centrifuge tube
• Add 1 ml of diluent (Reagent E )
• Add 200 μl of color developing solution (Reagent F )
• Vortex oscillation for 60 seconds
• Place in a mini tabletop centrifuge for 5 minutes at a speed of 1000g
• Carefully remove 1 ml of blue-green upper liquid phase into a new cuvette
• Immediately put into the spectrophotometer to detect: obtain absorbance readings
• Actual absorbance reading: sample absorbance reading - background absorbance reading
• The sample corresponds to the oleic acid concentration (mmol/L) according to the standard curve.

• Calculate sample activity

• Sample total activity and non-specific activity calculation

[According to the standard curve, the sample corresponds to the oleic acid concentration (mmol/L) X 10 (dilution factor) X 1 (reaction system capacity; ml)] ÷ [0.1 (sample capacity; ml) X 30 (reaction time; minute)] = micromol/ml/min ÷ (sample protein concentration) mg/ml = micromol/mg/min

• Sample specific activity calculation

Total sample activity - sample non-specific activity = sample specific activity

Precautions

• This product is 20 operations, including background control
• Wear gloves when handling
• Only 1 time for background determination during system operation
• Samples must be clarified, it is vital
• If the substrate solution (Reagent D) is viscous, it will be milky white after heating and can be used.
• After the colorimetric determination, the cuvette must be thoroughly cleaned.
• Sample readings above background reading indicate enzymatic activity
• It is recommended that the sample protein concentration to be tested be 50 μg/100 μl; if the sample enzyme activity is too low or too high, the sample size can be increased or decreased (the company provides Bradford Protein Concentration Quantitation Kit 30030.1)
• If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
• The lysosomal acid lipase unit activity is defined as the amount of enzyme required to release 1 micromole of free fatty acid per minute at 37 ° C, pH 5.0 as an active unit.
• The company provides a series of cell lipid enzyme detection reagent products

Quality Standard

• This product has been certified to be stable.
• This product has been identified and sensitive

Plant Protein

Plant-based protein powders are our hot selling products which can be widely used in sports nutrition and health&wellness fields. Among protein powder from different plant sources, rice protein, pea protein, sunflower seeds protein, pumpkin seeds protein is suitable for making protein bars, protein shakes. Take plant protein as subsititute of animal protein could reduce the risk of getting heart diseases, plant protein contain more fiber and do not contain less healthy compounds found in meat including saturated fat and cholesterol, plant protien are safe and vegan friendly choice.

Plant Protein,Protein Powder,Plant-Based Protein,Rice Protein

YT(Xi'an) Biochem Co., Ltd. , https://www.ytwholefood.com