Anti-Spastin antibody [Sp 6C6]

• IP

Collaborator

Immunoprecipitation - Anti-Spastin antibody [Sp 6C6] (AB77144)

Immunoprecipitation of SPAST in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1μ of ab77144 pre-coupled to Protein A beads. Samples were washed and processed for western blot with ab77144 at 1/500.

This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knock out cell lines.

All lanes:

Immunoprecipitation - Anti-Spastin antibody [Sp 6C6] (ab77144)

Predicted band size: 68 kDa

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• WB

Lab

Western blot - Anti-Spastin antibody [Sp 6C6] (AB77144)

Lanes 1-3 : Merged signal (red and green). Green - ab77144 observed at 52 kDa. Red - loading control ab181602 observed at 36 kDa.

ab77144 Anti-Spastin antibody [Sp 6C6] was shown to specifically react with Spastin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267238 (knockout cell lysate ab258698) was used. Wild-type and Spastin knockout samples were subjected to SDS-PAGE. ab77144 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Spastin antibody [Sp 6C6] (ab77144) at 1/500 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SPAST knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-spast-spastin-knockout-hek-293t-cell-line-ab267238'>ab267238</a>)

Lane 3:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 68 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-Spastin antibody [Sp 6C6] (AB77144)

ab77144 was shown to react with SPAST in wild-type HEK293T cells in Western blot with loss of signal observed in SPAST knockout cell line ab267238 (SPAST knockout cell lysate ab258698). Wild-type HEK293T and SPAST knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab77144 overnight at 4 °C at a 1/500 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2μg/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-Spastin antibody [Sp 6C6] (ab77144) at 1/500 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SPAST knockout HEK293T cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

SPAST knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

goat anti-rabbit HRP at 0.2 µg/mL

Predicted band size: 68 kDa

false

• WB

AbReview43580****

Western blot - Anti-Spastin antibody [Sp 6C6] (AB77144)

9% SDS-PAGE.

All lanes:

Western blot - Anti-Spastin antibody [Sp 6C6] (ab77144) at 1/1500 dilution

All lanes:

Human induced pluripotent stem cell derived neuron whole cell lysate at 20 µg

Secondary

All lanes:

HRP-conjugated goat anti-mouse IgG polyclonal at 1/10 dilution

Predicted band size: 68 kDa

Observed band size: 54 kDa,58 kDa,62 kDa,68 kDa

true

Exposure time: 12min

This image is courtesy of an anonymous Abreview