Anti-ATP6V1B2 antibody

6 product images

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  Immunoprecipitation - Anti-ATP6V1B2 antibody (ab73404)

  ATP6V1B2 was immunoprecipitated using 0.5mg Mouse Testis tissue lysate, 5µg of Rabbit polyclonal to ATP6V1B2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
  The antibody was incubated under agitation with Protein G beads for 10min, Mouse Testis tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
  Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab73404.
  Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).
  Band: 57kDa; ATP6V1B2

  All lanes: Immunoprecipitation - Anti-ATP6V1B2 antibody (ab73404)

  Predicted band size: 57 kDa

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  Western blot - Anti-ATP6V1B2 antibody (ab73404)

  Lanes 1-6 were blocked with 5% BSA, Lanes 7-12 were blocked with 3% milk.
  Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

  All lanes: Western blot - Anti-ATP6V1B2 antibody (ab73404) at 1 µg/mL

  Lanes 1 and 7: Hippocampus (Mouse) Tissue Lysate at 10 µg

  Lanes 2 and 8: Kidney (Mouse) Tissue Lysate at 10 µg

  Lanes 3 and 9: Testis (Mouse) Tissue Lysate at 10 µg

  Lanes 4 and 10: Human testis tissue lysate - total protein (ab30257) at 10 µg

  Lanes 5 and 11: Human kidney tissue lysate - total protein (ab30203) at 10 µg

  Lanes 6 and 12: Rat Hippocampus Tissue Lysate at 10 µg

  Secondary

  All lanes: Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

  Performed under reducing conditions.

  Predicted band size: 57 kDa

  Observed band size: 37 kDa, 57 kDa

  Exposure time: 30s

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  Immunocytochemistry/ Immunofluorescence - Anti-ATP6V1B2 antibody (ab73404)

  ICC/IF image of ab73404 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab73404, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) PC12 cells at 5µg/ml.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP6V1B2 antibody (ab73404)

  IHC image of ATP6V1B2 staining in human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab73404, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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  This image is courtesy of an anonymous customer review

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP6V1B2 antibody (ab73404)

  ab73404 staining RAt incisor tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2.5% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/1000 in 1% BSA/ 0.5% Triton X-100 in PBS) for 16 hours at 4°C. An undiluted peroxidase-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-ATP6V1B2 antibody (ab73404)

  ATP6V1B2 western blot using anti-ATP6V1B2 antibody ab73404. Publication image and figure legend from Sun, J., Li, Y., et al., 2022, Aging Cell, PubMed 34905649.

  
  

  ab73404 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab73404 please see the product overview.

  GDF11 decreases lysosomal function. (a) AML‐12 cells transient expressing GFP‐LC3 were cultured for 48 h in presence of GDF11 (100 ng/ml). After staining with LysoTracker Red DND‐99, cells were examined by confocal microscopy. Immunofluorescence confocal microscopy showing colocalization between LC3 puncta and LysoTracker Red with or without GDF11 (original magnification, 400×). (b) Quantification of the fluorescence intensity LC3 overlapping with Lysotracker signal normalized to vehicle controls. (c) Representative images of self‐quenched DQ‐BSA in AML‐12 cells (original magnification, 400×). (d) Quantification of the fluorescence intensity of DQ‐BSA normalized to vehicle controls. (e) Western blot analysis of m‐cathepsin B, ATP6V1a, and ATP6V1b2 protein expression. (f) Densitometry analysis of the m‐cathepsin B, ATP6V1a, and ATP6V1b2. The experiment was performed in triplicate with similar results. The data are shown as mean ± SD, *p < 0.05 compared to the vehicle group