Product Details
| abID | ab65352 |
|---|---|
| Cat # +Size | K332-100 |
| Size | 100 assays |
| Detection Method | Absorbance (440 nm) |
| Species Reactivity | Mammalian |
| Applications | Detects HAT activity by colorimetric method in mammalian samples |
| Features & Benefits | • Simple procedure • Fast and convenient • The detection can be continuous and suitable for kinetic studies |
| Kit Components | • 2X HAT Assay Buffer • HAT Substrate I • HAT Substrate II • NADH Generating Enzyme • HAT Reconstitution Buffer • Nuclear Extract 4 mg/ml |
| Storage Conditions | -20°C |
| Shipping Conditions | Dry Ice |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Histone acetyltransferases (HATs) have been implicated to play a crucial role in various cellular functions, such as gene transcription, differentiation, and proliferation. BioVision’s HAT Activity Colorimetric Assay Kit offers a convenient, nonradioactive system for a rapid and sensitive detection of HAT activity in mammalian samples. The kit utilizes active Nuclear Extract (NE) as a positive control and acetyl-CoA as a cofactor. Acetylation of peptide substrate by active HAT releases the free form of CoA which then serves as an essential coenzyme for producing NADH. NADH can easily be detected spectrophotometrically upon reacting with a soluble tetrazolium dye. The detection can be continuous and suitable for kinetic studies. The kit provides a simple, straightforward protocol for a complete assay.
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Which HATs these assays detect?
The assays detect eventually all HATs that can deacetylate histone.
DTT & Protease Inhibitor Interfere with the assay?
DTT or regular low dose of protease inhibitors should not interfere with the assay. High dose protease inhibitor may interfere the developer.
Which assays are more sensitive: Radioactive methods or your kits?
Radioactive detections are rather too complicated and involves in radioactive materials which are difficult to handle. We have not done direct comparison studies with the radioactive methods.
Can we use frozen tissues?
Yes, you can use frozen tissue. If you keep the tissue in -80C,it can stable for years (do not let the tissue dry. You need 10 to 20 mg of tissue to make lysate. The fluorometric assay is a little more sensitive (~4 FOLD) if you have a flourescence reader.
Can we run this experiment in a 1.5 ml cuvette?
Yes, but you need to scale up the quantities by 5 folds.
Can we use frozen samples 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.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you 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 this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Tzeng, Thing-Fong et al. (2016) Antioxidant-Rich Extract from Plantaginis Semen Ameliorates Diabetic Retinal Injury in a Streptozotocin-Induced Diabetic Rat Model, Nutrients. 2016 Sep 18;8(9).
Yang et al., PARP-1 Mediates LPS-Induced HMGB1 Release by Macrophages through Regulation of HMGB1 Acetylation.J. Immunol., Dec 2014; 193: 6114 - 6123.
Khalyfa et al., Sleep Fragmentation During Late Gestation Induces Metabolic Perturbations and Epigenetic Changes in Adiponectin Gene Expression in Male Adult Offspring Mice. Diabetes, Oct 2014; 63: 3230 - 3241.
Wang et al., Endothelial Cell Heparanase Taken Up by Cardiomyocytes Regulates Lipoprotein Lipase Transfer to the Coronary Lumen After Diabetes. Diabetes, Aug 2014; 63: 2643 - 2655.
Vecellio et al., The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients. Diabetes, Jun 2014; 63: 2132 – 2147.
For more citations of this product click here
