Bacterial ATP/ADP/AMP content high performance liquid chromatography (HPLC) quantitative detection kit instructions

High-performance liquid chromatography (HPLC) quantitative detection kit for bacterial ATP/ADP/AMP content

Product Manual (Chinese version)

The main purpose

Bacterial ATP/ADP/AMP content high performance liquid chromatography (HPLC) quantitative detection reagent is a kind of acid treatment by perchloric acid, after alkaline neutralization, under high performance liquid chromatography and ultraviolet spectrometer (254nm wavelength Detection and analysis, separation of ATP, ADP or AMP peaks to quantify the authoritative and classical technical method of adenine nucleotide content in samples. The technology has been carefully developed and successfully tested. It is suitable for the detection of ATP, ADP and AMP in various bacterial samples. The product is strictly sterile, ready to use, simple in operation and stable in performance.

technical background

Adenine nucleotides, adenosine, including adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate, are monomeric units that are components of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). And provide chemical energy to maintain the biochemical and physiological functions of tissue cell structures such as muscles and membranes in the energy metabolism pathway, regulate cell glycolysis, tricarboxylic acid cycle, electron transport system, and oxidative phosphorylation activities. Adenine nucleotides are low in concentration and unstable in cells, and their metabolism and energy status are usually evaluated by their different types, concentration differences, and distribution. Adenosine monophosphate (AMP), also known as 5'-adenosine nucleotide or 5'-adenylic acid, is a nucleotide found in (deoxy)ribonucleic acid. It is an ester of phosphoric acid and nucleoside adenosine and is composed of a phosphate group, a pentose nucleic acid sugar and a base adenine. The molecular formula is C10H14N5O7P, and the molecular weight is 347. AMP can be catalyzed by adenylate kinase to produce 2 molecules of ADP, or by ATP and ADP hydrolysis. AMP is often present in cells as adenosine-3',5'-cyclized monophosphate (cAMP). The ratio of AMP to ATP reflects the state of ATP production and the degree of fatty acid oxidation. Adenosine diphosphate (ADP), which participates in the ADP-ATP cycle, provides thermal energy conversion and homeostasis. Adenosine triphosphate (ATP) is present in all metabolically active cells and is a hallmark of cell viability. Once the cells are apoptotic or necrotic, the ATP concentration drops sharply. Acidic treatment with perchloric acid eliminates protein interference. After alkaline neutralization, ATP, ADP, and AMP peaks are separated and analyzed by high performance liquid chromatography and ultraviolet spectrometer (254 nm wavelength) to quantify the content.

product content

Acid solution (Reagent A) 5 ml

Neutral solution (Reagent B) 1 ml

Stream Liquid A (Reagent C) 500 ml

Stream Liquid B (Reagent D) 500 ml

Standard solution (Reagent E) 500 μl

Product manual 1 copy

storage method

The standard solution (Reagent E) is stored in a -20 ° C refrigerator, and the rest is stored in a 4 ° C refrigerator; the acidic liquid (Reagent A) and the neutralizing liquid (Reagent B) are corrosive, pay attention to the safety of operation;

User-supplied

1.5 ml centrifuge tube: container for sample handling

4°C (micro) benchtop centrifuge: for sample handling

High Performance Liquid Chromatography (HPLC): for quantitative determination

Experimental procedure

Sample preparation

• Prepare the sample of bacteria to be tested
• Perform bacterial counting: OD600 or direct counting on a spectrophotometer
• Transfer to pre-cooled 1.5 ml centrifuge tube
• Place in a 4°C mini tabletop centrifuge for 5 minutes at a speed of 10,000g (or 10000RPM, eg eppendorf 5415)
• Carefully remove the supernatant and retain the pellet
• Add 200 μl of pre-cooled acid solution (Reagent A )
• Vortex for 1 minute
• Transfer to a 1.5 ml centrifuge tube
• Place in a 4°C micro-tabletop centrifuge and centrifuge again for 10 minutes at a speed of 6000g.
• Carefully remove the supernatant into a new 1.5 ml centrifuge tube
• Add 31 μl of Neutral Solution (Reagent B ) and mix thoroughly
• Let it sit in the ice trough for 30 minutes
• Immediately transfer the supernatant to a new 1.5 ml centrifuge tube (note: avoid removing precipitated particles)
• Store in a -70 ° C refrigerator or in an ice trough to be tested

Second, measurement preparation

• Turn on the instrument for 15 minutes
• Set high performance liquid chromatography (HPLC) parameters according to the table below

• Running program

• Standard sample preparation

• Prepare 5 1.5 ml tubes, labeled 1 to 5
• Were added 100 microliters of a liquid phase stream A (Reagent C) to 1 to 5
• Pipette 100 μl of standard solution ( Reagent E ) into tube 1 and mix
• Carefully remove 100 μl of No. 1 tube diluted standard solution ( Reagent E ) into tube 2 and mix.
• Carefully remove 100 μl of No. 2 tube diluted standard solution ( Reagent E ) into tube No. 3 and mix well.
• Carefully remove 100 μl of No. 3 tube diluted standard solution ( Reagent E ) into tube 4 and mix.
• Put the 1 to 5 tubes into the ice tank for use, avoiding the light; the standard tube concentration is shown in the table below.

Fourth, chromatographic analysis

• Put the prepared standard solution (Reagent E) and the sample to be tested into the ice tank and wait
• 50 microliters per injection (per sample)
• Run 15 minutes each time (per sample) according to the test run procedure
• Obtain chromatographic peak map: peak area or peak, save record

V. Calculate sample ATP/ADP/AMP concentration

• Method 1: Calculate by software in the instrument
• Method 2: Construct a standard curve: the ordinate (Y-axis) is the peak area; the abscissa (X-axis) is the standard ATP/ADP/AMP concentration (micromol/L), and the sample corresponding ATP is obtained according to the standard curve. /ADP/AMP concentration (micromoles/liter)
• Method 3: Formula calculation (select a single concentration of standard solution)

Sample ATP/ADP/AMP concentration (micromol/L) = [sample peak area (X standard ATP/ADP/AMP concentration] ÷ Corresponding to the peak area of ​​the standard ATP/ADP/AMP concentration

Precautions

• This product is operated 25 times, including standard solution
• Wear gloves when handling
• Standard solution determination is only 1 time during system operation
• Reagents are volatile, pay attention to the closed cap or use the parafilm
• Acid solution (Reagent A) and neutralizer (Reagent B) are corrosive, pay attention to safe operation
• We recommend the use of liquid phase stream A (Reagent C) as a negative control or as a reference line
• Foreign matter residue, bubble generation, temperature imbalance, etc. of the column will cause abnormal peaks, time shifts, etc.
• The user can add a known concentration of standard solution (Reagent E ) as an internal reference standard to the sample, and then perform extraction to detect recovery (recovery).
• The retention time of adenine nucleotides is based on the actual peak of the standard solution (Reagent E ) used by the user.
• Users can calculate the actual concentration as required: nanomoles/ml, nanomoles/mg protein, etc.
• If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
• The reference map of adenine nucleotide HPLC is as follows:
• The company provides a series of adenine nucleotide detection reagent products

Quality Standard

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

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