HDM Fluorescent Kit

• Type:

  Application Kit

• Detection Method:

  Fluorometric

• Assay Principle:

  The DetectX® Demethylase Activity Kit is designed to quantitatively measure the enzymatic activity of formaldehyde-producing enzymes such as Histone Demethylases. The kit is unique in that the product of these enzymatic demethylation reactions, formaldehyde, is quantitated directly by a fluorescent product. No separation or washing is required. The kit has been validated for both LSD1 and JMJD2A Histone Demethylases (HDMs) . The kit provides optimized buffers for the HDMs LSD1 and JMJD2A, a stable formaldehyde standard, the Formaldehyde Detection Reagent (FDR) and two 96 well plates for detecting the generated fluorescent signal. The kit allows any enzymatic reaction generating formaldehyde to be measured. The end user will have to provide the demethylase system and any cofactors, etc. necessary for activity, along with any test inhibitors or activators. The kit allows end users to produce HDM activity in many in vivo and in vitro systems and then determine the activity by measuring formaldehyde generation. For in vitro studies, the HDM reaction should be carried out in our supplied buffers using optimized reaction conditions for the demethylation. For HDM samples in cell lysates, we include a specially formulated Cell Lysis Buffer, X050-100ML, that has been shown not to interfere with formaldehyde detection. Cell lysis buffers containing SDS and Triton X-100 inhibit the formaldehyde signal reaction and should not be used. Following the formaldehyde generating reaction, the reaction can be stopped by addition of a suitable inhibitor. The FDR is then added to all the wells. If calibration to formaldehyde is needed (for cross lab comparisons) then a formaldehyde standard curve generated from the supplied standard should be run. After a short incubation at 37C for 30 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 450 nm. The demethylase activity is determined based upon formaldehyde production, after making a suitable correction for any dilution of the sample, using software available with most fluorescence plate readers. We have provided two 96 well plates for measurement but this assay is adaptable for higher density plate formats. The end user should ensure that their HTS black plate is suitable for use with these reagents prior to running samples.

• Assay Protocol:

  Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

• Sample Type:

  LSD1- and Jumonji-type Demethylases

• Reconstitution:

  -20°C

• Components:

  Component Quantity Black Half Area 96 Well Plate 2 plates LSD1-type Assay Buffer 60 mL JMJD2A-type Assay Buffer 60 mL Formaldehyde Standard 500 uL DetectX® Formaldehyde Reagent 5 mL Demethylase Cell Lysis Buffer 100 mL Plate Sealers 2 Each Component Quantity Component Quantity Black Half Area 96 Well Plate 2 plates Black Half Area 96 Well Plate 2 plates LSD1-type Assay Buffer 60 mL LSD1-type Assay Buffer 60 mL JMJD2A-type Assay Buffer 60 mL JMJD2A-type Assay Buffer 60 mL Formaldehyde Standard 500 uL Formaldehyde Standard 500 uL DetectX® Formaldehyde Reagent 5 mL DetectX® Formaldehyde Reagent 5 mL Demethylase Cell Lysis Buffer 100 mL Demethylase Cell Lysis Buffer 100 mL Plate Sealers 2 Each Plate Sealers 2 Each

• NCBI Gene Symbol:

  HDM

• Gene Name URL:

  HDM