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Product Details
| Cat # +Size | K330-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em 350-380/440-460 nm) |
| Species Reactivity | Mammalian |
| Applications | Well suited for high throughput applications. HDAC inhibitors and substrates and related antibodies are also available separately. |
| Features & Benefits | • Simple two-step procedure; takes around than 1 hour • Fast and convenient • The assay method eliminates radioactivity/extractions/ and/or chromatography as used in the traditional assays. |
| Kit Components | • HDAC Substrate [Boc-Lys(Ac)-AMC, 4 mM] • 10X HDAC Assay Buffer • Lysine Developer • HDAC Inhibitor (Trichostatin A, 1 mM) • HeLa Nuclear Extract (5 mg/ml) • Deacetylated Standard [Boc-Lys-AMC, 4 mM] |
| Storage Conditions | -80°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Inhibition of histone deacetylase (HDAC) has been implicated to modulate transcription and induce apoptosis or differentiation in cancer cells. However, screening compounds for HDAC inhibition has been difficult due to the lack of convenient tools for analyzing HDAC activity. The Fluorometric HDAC Activity Assay Kit provides a fast and fluorescence-based method that eliminates radioactivity, extractions, or chromatography, as used in traditional assays. The new procedure requires only two easy steps, both performed on the same microtiter plate. First, the HDAC substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (e.g., HeLa nuclear extract). Deacetylation of the substrate sensitizes the substrate, so that further treatment with the Lysine Developer produces a fluorophore. The fluorophore can be easily analyzed using a fluorescence plate reader or a fluorometer. The assay is well suited for high throughput screening applications. HDAC inhibitors and antibodies are also available separately.
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Which HDAC kits among K330 and K331 is more sensitive?
K330-100 the Fluorometric Assay Kit is more sensitive (~5 times more sensitive than K331-100 the Colorimetric Assay Kit).
Which HDACs these assays detect?
The assays detect eventually all HDACs 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.
What are the sensitivities of the HDAC substrates?
Here are the sensitivities for our HDAC kits K330 and K331: K330-100 (Fluorometric): 0.5 - 10 µg of nuclear extract. K331-100 (Colorimetric): 10 – 200 µg of nuclear extract
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.
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.
How can I prepare cell lysate? Could you please share the method?
To make the cell lysate, you can scrape the cells off the plate and use a regular cell lysis buffer. For suspension cells, centrifuge and pellet the cells. Homogenize 1X10^6 cell in 100 µL of cell lysis buffer using a dounce homogenizer or sonicator on ice. Once lysis is completed, centrifuge the cells and use the supernatant for the assay. Please use a lysis buffer that does not contain strong denaturing detergents such as SDS and sodium deoxycholate. RIPA may be too harsh.
Notes:
Notes:
1. If you have to use enzymatic digestion such as use of trypsin for adherent cells, please wash the cell pellet with PBS (2X) before you prepare the lysate to remove any traces of trypsin.
2. Nuclear extract will have more HDAC activity than the cell lysate. So it is your choice to decide what kind of sample you would want to use for the assay.
Sylvie Berthier, A New Patient-Derived Metastatic Glioblastoma Cell Line: Characterisation and Response to Sodium Selenite Anticancer Agent. Cancers (Basel), Dec 2018; 30583471.
Zhang, Haiyue et al. (2017) Role of histone deacetylase expression levels and activity in the inflammatory responses of patients with chronic hepatitis B, Mol Med Rep. 2017 May;15(5):2744-2752.
Wheeler et al., The Autotaxin–Lysophosphatidic Acid Axis Modulates Histone Acetylation and Gene Expression during Oligodendrocyte Differentiation. J. Neurosci., Aug 2015; 35: 11399 - 11414.
Castonguay et al., Panspecies Small-Molecule Disruptors of Heterochromatin-Mediated Transcriptional Gene Silencing. Mol. Cell. Biol., Feb 2015; 35: 662 - 674.
Sudha Ananth et al., Induction of the Cystine/Glutamate Exchanger SLC7A11 in Retinal Pigment Epithelial Cells by the Antipsoriatic Drug Monomethylfumarate. Invest. Ophthalmol. Vis. Sci., Mar 2013; 54: 1592 - 1602.
For more citations of this product click here
