Immunofluorescent analysis of Caspase cleaved Htt (green) in 293T cells either transfected with an Htt23Q (left panel) or Htt148Q (right panel) stop construct ending in amino acid 513. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 5% normal goat serum for 15 minutes at room temperature. Cells were probed with an Htt neo-epitope 513 polyclonal antibody (Product# PA1-002) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with goat-anti-rabbit secondary antibody at room temperature. Nuclei (blue) were stained with Hoechst dye.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide conjugated to KLH via cysteine corresponding to residues HTLQADSVD (505-513) of Human HTT.|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||1:50 - 1:200|
|Immunohistochemistry (IHC)||1:50 - 1:200|
|Western Blot (WB)||1:500|
* 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.
|Western Blot (WB)||See 1 publications below|
PA1-002 has been successfully used in ELISA, Western Blot, immunofluorescence, and immunohistochemistry applications with 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 PA1-002 recognizes the 513 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.
Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease.
PA1-002 was used in western blot to validate the role of caspase activity in Huntington's Disease
|Hermel E,Gafni J,Propp SS,Leavitt BR,Wellington CL,Young JE,Hackam AS,Logvinova AV,Peel AL,Chen SF,Hook V,Singaraja R,Krajewski S,Goldsmith PC,Ellerby HM,Hayden MR,Bredesen DE,Ellerby LM||Cell death and differentiation (11:424)||2004|