乙酸检测试剂盒[乙酸激酶法] Acetic Acid (Acetate Kinase Manual Format) 货号:K-ACETRM Megazyme中文站

乙酸检测试剂盒[乙酸激酶法]

英文名:Acetic Acid (Acetate Kinase Manual Format)

货号:K-ACETRM

规格:72 assays (manual) / 720 assays (microplate)

分析物意义: 常见食品的组分

Megazyme检测试剂盒优点: K-ACETRM 是运用AK和磷酸乙酰转移酶的新型、快速的手工检测试剂盒。试剂稳定

K-ACETAK (自动) 是一种以乙酸激酶(AK)为基础的,新型、稳定、快速的检测试剂盒,具有良好的线性。

This rapid and reliable manual acetic acid kit is simple to perform (only two absorbance readings required), and because a true end-point is measured, does not involve complicated calculations like other kits. This product is very stable both during storage and use (> 2 years), has extended linearity (compared to ACS based kits), contains PVP to prevent tannin inhibition, and is performed at a relatively low pH (7.4), thus minimising ester hydrolysis related interference. This method is suitable for the measurement of acetic acid/acetate in foods, beverages and other materials. Content:72 assays

乙酸检测试剂盒[乙酸激酶法]

Manual format UV-method for the determination of Acetic Acid 
in foodstuffs, beverages and other materials

Principle:
                         (acetate kinase)
(1) Acetic acid + ATP → acetyl-phosphate + ADP

                          (phosphotransacetylase)
(2) Acetyl-phosphate + CoA → acetyl-CoA + Pi

           (pyruvate kinase)
(3) ADP + PEP → ATP + pyruvate

                       (D-lactate dehydrogenase)
(4) Pyruvate + NADH + H+ → D-lactic acid + NAD+

Kit size:                            72 assays (manual) / 720 (microplate)
Method:                            Spectrophotometric at 340 nm
Reaction time:                  ~ 4 min
Detection limit:                 0.063 mg/L
Application examples: 
Wine, beer, fruit and fruit juices, soft drinks, vinegar, vegetables, 
pickles, dairy products (e.g. cheese), meat, fish, bread, bakery products
(and baking agents), ketchup, soy sauce, mayonnaise, dressings, paper
(and cardboard), tea, pharmaceuticals (e.g. infusion solutions), feed
and other materials (e.g. biological cultures, samples, etc.)
Method recognition:         Improved method

Advantages

  • Improved assay format (only two absorbance readings required)
     
  • All reagents stable for > 2 years after preparation
     
  • PVP incorporated to prevent tannin inhibition
     
  • Very rapid reaction (~ 4 min)
     
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
     
  • Very competitive price (cost per test)
     
  • Suitable for Manual and Microplate formats

Q1. Is the acetic acid kit specific for acetate?

Propionate may react more slowly than acetate.

Q2. Is the K-ACETRM Assay Kit suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed but dilution (if required) in distilled water. 

Q3. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q4. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q5. Does the decolourising preparation remove some VA during the process?

No, however the sample preparation process can be tested by adding a known amount of acetic acid standard and assessing the recovery of this. 

Q6. Can acetic acid be measured in culture/fermentation media?

Acetic acid in liquid cell culture media/supernatants or fermentation samples can be determined without any sample treatment (except clarification by centrifugation or filtration) and appropriate dilution in distilled water. 

Q7. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme ([email protected]). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q8. What are the major the differences between the various acetic acid test kits?

Megazyme produces 4 acetic acid test kits:
K-ACET: uses the traditional ACS reaction.  Manual format for use with spectrophotometers.
K-ACETAF: uses the traditional ACS reaction.  Automated format for use with auto-analysers.
K-ACETAK: uses the more recently developed and more rapid acetate kinase reaction.  Automated format for use with auto-analysers.
K-ACETRM: uses the more recently developed and more rapid acetate kinase reaction.  Manual format for use with spectrophotometers. 

Q9. Which acetic acid kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore K-ACETAK or K-ACETAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-ACET or K-ACETRM may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate.  However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.  Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a     mean conversion factor between the cuvette values and the microplate values.


Acetic Acid Kit Recommendation For Microplate Format:
Either K-ACETRM or K-ACETAK is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-ACETRM:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3. 

Q10. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q11. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q12. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q13. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q14. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q15. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.