Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab157220).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

This data was developed using ab110638, the same antibody clone in a different buffer formulation.

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)] - BSA and Azide free (AB240141)

This data was developed using ab157220, the same antibody clone in a different buffer formulation.

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)] (<a href='/en-us/products/primary-antibodies/vps35-antibody-epr11501b-ab157220'>ab157220</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

VPS35 knock-out HAP1 lysate at 20 µg

false

• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

This data was developed using ab157220, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (<a href='/en-us/products/primary-antibodies/vps35-antibody-epr11501b-ab157220'>ab157220</a>) 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

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

This data was developed using ab157220, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-VPS35 antibody [EPR11501(B)] (<a href='/en-us/products/primary-antibodies/vps35-antibody-epr11501b-ab157220'>ab157220</a>) 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

false

Exposure time: 48s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab157220).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab157220).

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-VPS35 antibody [EPR11501(B)] - BSA and Azide free (AB240141)

Intracellular flow cytometric analysis of permeabilized MOLT4 cells labeling VPS35 with ab157220 at 1/10 dilution (red) or a rabbit IgG (negative) (green). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab157220).