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The maintenance of organelles in eukaryotic cells depends on sorting proteins, which ensure the proper delivery of organelle-specific proteins. In S. cerevisiae, VPS35 has a direct role in the retrieval of vacuolar cargo proteins, suggesting that human VPS35 may have an analogous function in the maintenance of lysosomes.
Our Abpromise guarantee covers the use of ab10099 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.1 - 0.3 µg/ml. Detects a band of approximately 85 kDa (predicted molecular weight: 91 kDa).Can be blocked with Human VPS35 peptide (ab23181).|
|ICC/IF||Use at an assay dependent concentration.|
Detected by chemiluminescence.
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: VPS35 knockout HAP1 cell lysate (20 µg)
Lane 3: NIH3T3 cell lysate (20 µg)
Lane 4: A549 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab10099 observed at 85 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab10099 was shown to specifically react with VPS35 when VPS35 knockout samples were used. Wild-type and VPS35 knockout samples were subjected to SDS-PAGE. ab10099 and ab181602 (loading control to GAPDH) were diluted to 1 µg/ml and 1/1000 respectively and incubated overnight at 4°C. Blots were developed with IRDye® 800CW Donkey anti-Goat IgG (H + L) and Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216779) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
Incubated with the primary antibody for 1 hour.
Immunocytochemistry/Immunofluorescence analysis of PFA-fixed and saponin permeabilized HEK293 cells labelling VPS35 with ab10099.
Immunocytochemistry/Immunofluorescence analysis of PFA-fixed and saponin permeabilized SHSY5Y cells labelling VPS35 with ab10099.