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Product Details
| Cat # +Size | K752-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm) |
| Species Reactivity | Mammalian |
| Applications | Evaluation of pentose phosphate pathway |
| Features & Benefits | • Simple procedure • Fast and convenient • Sensitive assays for measuring ALT activity in various biological samples and the kit is also suitable for high throughput analyses. |
| Kit Components | • ALT Assay Buffer • OxiRed Probe (in DMSO) • ALT Enzyme Mix • ALT Substrate • Pyruvate Standard (100 nmol/µl) • ALT positive control |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Alanine transaminase (ALT) is a transaminase enzyme (EC 2.6.1.2). It is also called serum glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT). ALT is found in serum and in various bodily tissues, but is usually associated with the liver. It catalyzes the reaction: α-ketoglutarate + alanine ⇌ glutamate + pyruvate It is commonly measured clinically as a part of a diagnostic liver function test to determine liver health. Diagnostically, it is almost always measured in units/liter (U/L). In BioVision’s ALT Assay Kit, ALT catalyzes the transfer of an amino group from alanine to ⍺-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate. The pyruvate is detected in a reaction that concomitantly converts a nearly colorless probe to both color (λmax = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The kit provides a rapid, simple, sensitive, and reliable test suitable for high throughput activity assay of ALT with a detection limit of 10 mU per well.
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If I am measuring LDH alpha (LDH5), which converts pyruvate to lactate, will this assay work for me?
Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant inter-conversion of NADH and NAD+. It converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver. So your LDH5 converts pyruvate to lactate and vice versa. In our assay, NAD (produced during pyruvate to lactate conversion by LDH5) is reduced to NADH, which interacts with a probe to produce a color (max = 450 nm). In essence, this assay can work for your objective. The reaction conditions and the relative amount of pyruvate and lactate decide which direction the reaction will go.
Does phenol red in the edia affect this assay?
Since 2-50 ul sample is added per well, the color from phenol red is diluted by the assay buffer/reaction mix. Typically this does not affect the assay. The OD 450 readings are in the yellow.
How can adherent cells be used for this assay?
Trypsinization can be used to detach cells. Then collect the cells, spin down, remove medium+trypsin and then proceed with homogenization using the assay buffer in the kit. It is important to be careful since over-trypsinization might damage the plasma membrane and leak LDH into the medium.
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.
Is it essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, it is strongly recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Is the assay specific in a way that can discriminate between pyruvate formation from Pyruvate Kinase (and other pyruvate synthesizing enzymes) and Pyruvate from ALT?
The kit cannot distinguish between pyruvate formed from Pyruvate Kinase and ALT. It measures all Pyruvate. However, since our reaction mix favors the ALT reaction, we believe that the endogenous pyruvate will be negligible. If your samples contain Pyruvate, you can include a background control, where you do not the substrate to the reaction mix. The signal that you measure will be generated by endogenous pyruvate.
Will the ALT Assay Buffer disrupt mitochondria? Could you please tell me what kind of buffer is the ALT buffer? Will it solubilize mitochondria? This is important for us because GPT2 from mitochondria could interfere with the results.
No, theoretically it should not lyse the mitochondrial membrane. Please homogenize the sample gently. The Assay Buffer is a mix of different salts and detergent.
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