Western blot quantification4/25/2023 The different domains of the OPA1 protein are involved in distinct functions. To support cristae biogenesis and cristae junction maintenance, OPA1 interacts with the mitochondrial contact site and cristae organizing system (MICOS) complex ( 20, 21). OPA1 facilitates IMM fusion by means of a GTP-coupled reaction and interaction with cardiolipin ( 18, 19). Originally linked to IMM fusion ( 15), OPA1 plays additional roles in cristae maintenance ( 16) and mitochondrial fission ( 17). Splice sites 1 and 2 are relevant for protein processing and lead to long and short forms of OPA1 ( 13, 14). OPA1 is a nuclear gene that encodes eight different isoforms (gene ID: 4876). Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion yet, their specific roles remain elusive. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. This means you can use it to adjust values for your other antibody of interest (which also should be in it's linear range of detection.).Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Thus, you know that your antibody will produce a proportionally stronger signal intensity if you stay within this range of protein amount loaded on the gel. However, there is a roughly linear relationship if you limit the curve to the samples with less material loaded. I've taken the data from the 28th that you posted here, and plotted it for you, and as you can see, there is a non-linear relationship between the amount sample loaded and the signal intensity you measure. I am unsure though, whether or not this would solve your problem, which you actually already have solved yourself. Your antibody dilution will of course depend on its initial concentration and the affinity/avidity for the antigen, and it is entirely possible that you would be able to use less antibody. There could be various reasons for a non-linear behaviour for your antibody, such as molecular crowding, saturation of your membrane or incomplete transfer from your gel.
0 Comments
Leave a Reply.AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |