• Detection method- Absorbance (412 nm)
• Sample type- Cell and Tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids etc.
• Species reactivity- Mammalian
Features & Benefits:
• Simple procedure
• Fast and convenient
• Sensitive assays for measuring MPO as low as 0.05 mU per well
• MPO Assay Buffer
• DTNB Probe
• MPO Substrate
• Stop Mix
• MPO Positive Control
Myeloperoxidase (MPO) is a peroxidase enzyme (EC 220.127.116.11) most abundantly expressed in neutrophil granulocytes. It is a lysosomal protein stored in the azurophilic granules of the neutrophil. MPO contains a heme pigment which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus. MPO catalyzes the production of hypochlorous acid (HClO) from hydrogen peroxide (H₂O₂) and chloride anion (Cl-, or the equivalent from a non-chlorine halide). MPO also oxidizes tyrosine to a tyrosyl radical using hydrogen peroxide as an oxidizing agent. In BioVision’s MPO Assay Kit, the HClO produced from H₂O₂ and Cl- is reacted with taurine to generate the taurine chloramine, which subsequently reacts with the TNB2- probe to eliminate color (λ = 412 nm). The kit provides a rapid, simple, sensitive, and reliable test suitable for high throughput activity assay of MPO. This kit can be used to detect MPO as low as 0.05 mU per well.
USAGE: For Research Use Only! Not For Use in Humans.
How do you recommend standardizing the amount of sample used for all the wells?
Start with the same weight of tissue and homogenize in 2-3 volumes of the assay buffer. Then a total protein quantitation assay can be done and the same total protein can be added to each well for all samples. If a protein assay is not done, the same sample volume shoul dbe used or all samples.
The same customer used K744 and K745 but got very different raw data for the increasing dilutions of the samples. Why?
It is very important to be able to distinguish the two kits by principle. For K744, the lower the OD, the higher the MPO activity. If you add too much sample, the OD will be so low; it could be below the detection limit of absorbance instruments. For K745, the higher the RFU, the higher the MPO activity. So, for this kit, adding too much sample can saturate the detector and the substrate can be limiting. This will result in discrepant differences between dilutions.
The OD values from the samples are very high when 400 ul buffer is used with 10 mg tissue. What should we do?
Very high sample OD values means there is no or negligible MPO activity in the samples. The lower the OD the higher the MPO activity in this assay. It seems 400 ul buffer for 10 mg tissue might be too much, making the sample very dilute. You might either need to use less buffer or use more sample in the assay.Another point is that the starting OD for all samples should be similar and gradually the OD will get lower and lower as MPO activity proceeds over time.
How much cells/tissue is needed for this assay?
There is no set amount of cells/tissue needed for this assay. The amount depends on how much active MPO is present in the samples. Typically 1-2 million cells and 10-100 mg tissue can be used per assay.
What is the activity of the positive control? How can the value be higher to compare with samples?
The positive control is only a benchmark sample. As long as the values are within the range of the std curve this is fine. The positive control is not be used to compare values with the samples. The positive control is provided to validate that the assay components are all working. If the values are low, the customer can add more volume to get higher values but this is not necessary as long as the values are within the std. curve range. MPO is a very vulnerable enzyme to freeze-thaw and can lose activity with storage over time.
What is the dilution factor used for?
If a certain volume of neat sample is added to the well and volume is made up with the assay buffer up to 50 ul, then dilution factor does not apply. If the sample is prediluted before adding to the well, then the dilution factor is used. For example, if 10 ul of a 5x diluted sample is used, then V=0.01 ml and Dilution factor =5.
If 25 ul of test sample is added in each well what how much and what solution should be used for parallel background control - Water/Assay buffer?
The background control is to account for any engogeous sample-related interference and hence if you are adding 25 ul of sample, you should add the same volume of each sample in a parallel well for the control.
Can one background control be used for all samples?
Ideally, it is recommended to have parallel controls for each sample so that corresponding background can be subtracted for each sample correctly.
Can this kit be used with frozen cells/tissue?
We always prefer fresh samples for the most accurate results. It is possible to use frozen cells/tissue samples as long as they have been stored with minimum delay after collection and have been stored at -80C without freeze-thaw. However, the MPO enzyme is quite vulnerable to losing activity during storage. The functional enzyme contains disulphide bonds and if reduced, the enzyme can lose activity. It also contains heme which can get oxidized by the peroxide generated by MPO activity and the result is that the enzyme loses activity by auto-oxidation.
What is the shelf-life of the kit? What about the positive control is stored unthawed at -20C or -80C for 2-3 years?
The shelf-life of the unopened kit is 1 year from the date of shipping from us when stored at -20C. Trypsin is notoriously well known for its self-destructive protease activity when stored for long periods of time. Once reconstituted, the positive control is to be used within 2months for best results. Hence we do not expect the positive control enzyme to retain normal activity over 2-3 years even at -20C or -80C.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.