Analysis Note

Control501 Mel human melanoma cells, wild-type human, rat, mouse osteoclast cells.

Application

Immunoprecipitation:
A previous lot of this antibody was used in IP. (2 µg/mg of protein lysate)

Gel supershift assays:
A previous lot of this antibody was used in EMSA.

Immunohistochemistry(paraffin):
A previous lot of this antibody was used in IH on frozen and formalin/paraffin tissue sections.

Optimal working dilutions must be determined by end user.

Anti-Microphthalmia (Mi) Antibody, clone C5 is a Mouse Monoclonal Antibody for detection of Microphthalmia (Mi) also known as Microphthalmia-associated transcription factor & has been tested in EMSA, IHC, IHC(P), IP & WB.

General description

MiTF (Microphthalmia associated transcription factor) is a basic helix loop helix leucine zipper (b HLH ZIP) transcription factor implicated in pigmentation, mast cells and bone development. Mutations in MiTF cause auditory pigmentary syndromes, such as Waardenburg syndrome type II, type IIa and Tietz syndrome in humans. There are two known isoforms of MiTF differing by 66 amino acids at the NH2 terminus. Shorter forms are expressed in melanocytes and run as two bands at 52 kDa and 56 kDa, while the longer Mi form runs as a cluster of bands at 60-70 kDa in osteoclasts and in B16 melonoma cells (but not other melanoma cell lines), as well as mast cells and heart. MiTF plays a critical role in the differentiation of various cell types as neural crest-derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium. Mi is a basic helix-loop-helix-leucin zipper (b-HLH-ZIP) transtripotion factor implicated in pigmentation, mast cells and bone development. The mutation of Mi causes Waardenburg Syndrome type II in humans. In mice, a profound loss of pigmented cells in the skin eye and inner ear results, as well as osteopetrosis and defects in natural killer and mast cells. These melanocyte isoforms have been shown by two dimensional tryptic mapping to differ in c-Kit-induced phosphorylation. Osteopetrotic rat strain harbors a large genomic deletion encompassing the 3′ half of Mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts.

Immunogen

Hybridoma produced by the fusion of splenocytes from RBF/DnJ mice immunized with an N-terminal fragment of human microphthalmia protein and mouse myeloma NS1 cells.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Format: Purified

Purified mouse monoclonal IgG1 in phosphate buffered saline with 0.08% sodium azide.

Quality

Routinely evaluated by Western Blot on Mouse Brain lysates.

Western Blot Analysis:
1:500 dilution of this lot detected Mi on 10 µg of Mouse Brain lysates.

Specificity

In Western blotting, it recognizes a doublet of 52-56kDa, identified as serine-phosphorylated and unphosphorylated forms of melanocytic isoforms of microphthalmia (Mi). There are two known isoforms of Mi differing by 66 amino acids at the NH2 terminus. Shorter forms are expressed in melanocytes and run as two bands at 52kDa and 56kDa, while the longer Mi form runs as a cluster of bands at 60-70kDa in osteoclasts and in B16 melonoma cells (but not other melanoma cell lines), as well as mast cells andheart. It reacts with both melanocytic as well as the nonmelanocytic isoforms of Mi. This Ab does not cross-react with other b-HLH-ZIP factors by DNA mobility shift assay.

Storage and Stability

Stable for 1 year at -20°C in undiluted aliquots from date of receipt.
Handling Recommendations: Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage the IgG1 and affect product performance.

Target description

53-56 kDa