The Mouse Microphthalmia-Associated Transcription Factor (MITF) ELISA Kit is a highly sensitive and specific assay designed for the accurate quantification of MITF levels in mouse serum, plasma, and cell culture supernatants. This kit is optimized for reliable and reproducible results, making it perfect for a variety of research applications.MITF is a key transcription factor that plays a crucial role in the regulation of melanocyte development, function, and differentiation. Dysregulation of MITF has been linked to various diseases, including melanoma and certain genetic conditions.
Therefore, the measurement of MITF levels using this ELISA kit can provide valuable insights into these diseases and aid in the development of potential therapeutic interventions.Overall, the Mouse MITF ELISA Kit is a valuable tool for researchers looking to study the role of MITF in various biological processes and disease states. Its high sensitivity and specificity ensure accurate results, making it a must-have for any laboratory working in the field of molecular biology and genetics.
Transcription factor that regulates the expression of genes with essential roles in cell differentiation, proliferation and survival. Binds to M-boxes (5'-TCATGTG-3') and symmetrical DNA sequences (E-boxes) (5'-CACGTG-3') found in the promoters of target genes, such as BCL2 and tyrosinase (TYR). Plays an important role in melanocyte development by regulating the expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1). Plays a critical role in the differentiation of various cell types, such as neural crest-derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium.
Uniprot:
Q08874
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Microphthalmia-associated transcription factor
Sub Unit:
Efficient DNA binding requires dimerization with another bHLH protein. Binds DNA in the form of homodimer or heterodimer with either TFE3, TFEB or TFEC. Identified in a complex with HINT1 and CTNNB1 (By similarity). Interacts with KARS (PubMed:14975237).
Research Area:
Immunology
Subcellular Location:
Nucleus
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
MITF: a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. Plays a critical role in the differentiation of various cell types including neural crest- derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium. Two isoforms are known: the M-isoform is expressed exclusively in melanocytes, while the A-isoform has a much broader range of expression. Mutations in MITF can lead to Waardenburg syndrome. Ten alternatively spliced isoforms have been described.Protein type: DNA-binding; Transcription factor; OncoproteinChromosomal Location of Human Ortholog: 3p14.2-p14.1Cellular Component: protein complex; nucleusMolecular Function: protein dimerization activity; RNA polymerase II transcription factor activity, enhancer binding; protein binding; chromatin bindingBiological Process: transcription from RNA polymerase II promoter; cell fate commitment; camera-type eye development; regulation of osteoclast differentiation; regulation of transcription, DNA-dependent; positive regulation of transcription, DNA-dependent; melanocyte differentiation; protein complex assembly; positive regulation of transcription from RNA polymerase II promoter; osteoclast differentiation; negative regulation of transcription from RNA polymerase II promoter; bone remodeling; regulation of cell proliferationDisease: Albinism, Ocular, With Sensorineural Deafness; Waardenburg Syndrome, Type 2a; Melanoma, Cutaneous Malignant, Susceptibility To, 8; Tietz Syndrome
UniProt Protein Details:
NCBI Summary:
This gene encodes a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. It regulates the differentiation and development of melanocytes retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Heterozygous mutations in the this gene cause auditory-pigmentary syndromes, such as Waardenburg syndrome type 2 and Tietz syndrome. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
microphthalmia-associated transcription factor; class E basic helix-loop-helix protein 32
UniProt Protein Name:
Microphthalmia-associated transcription factor
UniProt Synonym Protein Names:
Class E basic helix-loop-helix protein 32
Protein Family:
Microphthalmia-associated transcription factor
UniProt Gene Name:
MITF
UniProt Entry Name:
MITF_HUMAN
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
4°C
Plate Sealer
5
-
Other materials and equipment required:
Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
