Anti-VPS35 antibody [EPR11501(B)]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labeling VPS35 with ab157220 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

ab157220 staining VPS35 in wild-type HAP1 cells (top panel) and VPS35 knockout HAP1 cells (bottom panel). The cells were fixed with 100% Methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab157220 at 0.2 µg/mL and ab7291 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in pseudo colour green) and goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) (shown in pseudo colour red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in pseudo colour blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling VPS35 with ab157220 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab157220 Anti-VPS35 antibody [EPR11501(B)] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

ab157220 was shown to react with VPS35 in wild-type HAP1 cells in Western blot with loss of signal observed in a VPS35 knockout cell line. Wild-type HAP1 and VPS35 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab157220 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (ab157220) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

VPS35 knock-out HAP1 lysate at 20 µg

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• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (ab157220) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 92 kDa

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Exposure time: 48s

• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (ab157220) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat heart tissue lysate at 20 µg

Lane 3:

Rat kidney tissue lysate at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 92 kDa

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Exposure time: 26s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

ab157220 staining VPS35 in wild-type HAP1 cells (top panel) and VPS35 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab157220 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Intracellular flow cytometric analysis of permeabilized MOLT4 cells labeling VPS35 with ab157220 at 1/10 dilution (red) or a rabbit IgG (negative) (green).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling VPS35 with ab157220 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : VPS35 knockout HAP1 cell lysate (20 μg)
Lane 3 : NIH/3T3 cell lysate (20 μg)
Lane 4 : A549 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab157220 observed at 91 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab157220 was shown to specifically react with VPS35 in wild-type HAP1 cells. No band was observed when VPS35 knockout samples were examined. Wild-type and VPS35 knockout samples were subjected to SDS-PAGE. ab157220 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (ab157220)

Predicted band size: 92 kDa

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• WB

Unknown

Western blot - Anti-VPS35 antibody [EPR11501(B)] (AB157220)

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (ab157220) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

Raji cell lysate at 10 µg

Lane 4:

MOLT4 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 92 kDa

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