Western blot analysis of over-expressed recombinant HTT fragment lysates was performed by loading 20 µg of lysate per well onto a 4-12% Bis-Tris polyacrylamide gel. Lysate transfected with empty PTET vector was loaded as negative control (Lane 2). Proteins were transferred to a nitrocellulose membrane and blocked with 3% BSA/TBST for at least 1 hour. Membranes were then probed with a neoepitope-specific mouse monoclonal antibody (Product # MA1-058) at a dilution of 1:500 overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-HRP secondary antibody at 1:30,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Mouse / IgG|
|Immunogen||Synthetic peptide conjugated to KLH via cysteine corresponding to residues CSDPAMDLND (544-552) of Human HTT.|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||2.5 µg/ml|
|Immunohistochemistry (IHC)||2.5 µg/ml|
|Western Blot (WB)||1:500 - 1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
MA1-058 has been successfully used in Western blot, immunofluorescence, and immunohistochemistry on human and mouse samples.
Neoepitope antibodies distinguish smaller cleaved fragments or processed forms of proteins versus the intact full-length or precursor by using a designed peptide purification process to maximize immunoreactivity to a specific cleavage site.
Human HTT caspase cleavage sites generate fragment-specific forms of the protein. Caspase-3/7 has been shown to generate cleavage sites at animo acids 513 and 552. Caspase-2 cleaves at amino acid 552 and caspase-6 at amino acid 586. Neo-specific antibody MA1-058 recognizes the 552 cleaved fragment without detecting the full-length form.
Huntingtin is a disease gene linked to Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. This is thought to be caused by an expanded, unstable trinucleotide repeat in the huntingtin gene, which translates as a polyglutamine repeat in the protein product. The huntingtin locus is large, spanning 180 kb and consisting of 67 exons. The huntingtin gene is widely expressed and is required for normal development. It is expressed as 2 alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues. The larger transcript is approximately 13.7 kb and is expressed predominantly in adult and fetal brain whereas the smaller transcript of approximately 10.3 kb is more widely expressed. The genetic defect leading to Huntington's disease may not necessarily eliminate transcription, but may confer a new property on the mRNA or alter the function of the protein.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.