Anti-SNX27 antibody [1C6]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SNX27 antibody [1C6] (AB77799)

Overlay histogram showing Jurkat cells stained with ab77799 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab77799, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SNX27 antibody [1C6] (AB77799)

ab77799 staining SNX27 in Jurkat cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 overnight at 4°C with ab77799 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IP

Unknown

Immunoprecipitation - Anti-SNX27 antibody [1C6] (AB77799)

SNX27 was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Mouse monoclonal to SNX27 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, Hek293 whole cell extract 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 ab77799.
Secondary : Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band : 61kDa : SNX27.

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Immunoprecipitation - Anti-SNX27 antibody [1C6] (ab77799)

Predicted band size: 61 kDa

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• WB

Project

Western blot - Anti-SNX27 antibody [1C6] (AB77799)

Blocking : 5% Milk in TBS-T.

Two bands are observed very close together, at around 60-kDa. There are four reported isoforms of the SNX27 protein (Swissprot). We hypothesise that this antibody detects both isoform 1 and isoform 2, which are 61-kDa and 60-kDa respectively. As the immunogen is derived from the N-terminal region of SNX27, the antibody is not expected to react with isoforms 3 or 4.

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Western blot - Anti-SNX27 antibody [1C6] (ab77799) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 4:

A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate at 10 µg

Lane 5:

MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

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Exposure time: 15min

• WB

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Western blot - Anti-SNX27 antibody [1C6] (AB77799)

4-12% bis-tris gel, MES buffer

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Western blot - Anti-SNX27 antibody [1C6] (ab77799) at 1/1000 dilution

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human kidney whole cell lysate at 30 µg

Secondary

All lanes:

IRDye® 680RD Goat anti-Mouse IgG (H + L) at 1/15000 dilution

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Exposure time: 5min

This image is courtesy of an anonymous Abreview