Anti-Spastin antibody [Sp 3G11/1]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Spastin antibody [Sp 3G11/1] (AB31850)

IHC image of Spastin staining in Human lung adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab31850, 10μg/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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IP

Characteriser

Immunoprecipitation - Anti-Spastin antibody [Sp 3G11/1] (AB31850)

Immunoprecipitation of SPAST in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1μg of ab31850 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 3G11/1] (ab31850)

Predicted band size: 68 kDa

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• WB

Lab

Western blot - Anti-Spastin antibody [Sp 3G11/1] (AB31850)

False colour image of Western blot : Anti-Spastin antibody [Sp 3G11/1] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab31850 was shown to bind specifically to Spastin. A band was observed at 55 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SPAST knockout cell line ab267238 (knockout cell lysate ab258698). To generate this image, wild-type and SPAST knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850) at 1/500 dilution

Lane 1:

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

Lane 2:

Spastin knockout HEK-293T 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:

SH-SY5Y cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 68 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-Spastin antibody [Sp 3G11/1] (AB31850)

ab31850 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 ab31850 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 3G11/1] (ab31850) 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:

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

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• WB

Unknown

Western blot - Anti-Spastin antibody [Sp 3G11/1] (AB31850)

There are two splice isoforms of spastin (one without exon4) and two alternative ATG start sites, which may determine the localisation of the translate protein. 52kDa is the major band.

All lanes:

Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850) at 1/500 dilution

All lanes:

rat brain synaptosome

Predicted band size: 68 kDa

Observed band size: 52 kDa

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