Anti-USP9x antibody

• ICC

Lab

Immunocytochemistry - Anti-USP9x antibody (AB19879)

ab19879 staining USP9x in Hek293 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab19879 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed 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 red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IP

Unknown

Immunoprecipitation - Anti-USP9x antibody (AB19879)

USP9x was immunoprecipitated using 0.5mg Caco2 whole cell extract, 5µg of Rabbit polyclonal to USP9x 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, Caco2 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 ab19879.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 290kDa : USP9x.

All lanes:

Immunoprecipitation - Anti-USP9x antibody (ab19879)

Predicted band size: 292 kDa

false

• IHC (PFA fixed)

Collaborator

Immunohistochemistry (PFA fixed) - Anti-USP9x antibody (AB19879)

Immuofluorescent staining for USP9X in the rat hippocampus (dentate gyrus) using ab19879 (1/300 = 0.07μg/ml). Image is taken with X10 objective. ab19879 was incubated overnight at RT. Secondary antibody used was anti-rabbit Alexa fluor 488 (1/1000 for 2h at RT). Rats were intracardially perfused with paraformaldehyde 4%, brain tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30μm coronal sections were cut on a cryostat for free floating IHC.

This image is courtesy of Sophie Pezet, King's College London, United Kingdom

• WB

Lab

Western blot - Anti-USP9x antibody (AB19879)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : USP9x knockout HAP1 cell lysate (20 μg)
Lane 3 : T84 cell lysate (20 μg)
Lane 4 : NIH3T3 cell lysate (20 μg)
Lanes 1 to 4 : Merged signal (red and green). Green - ab19879 observed at 290 kDa. Red - loading control, ab181602 observed at 124 kDa.
ab19879 was shown to specifically react with USP9x when USP9x knockout samples were used. Wild-type and USP9x knockout samples were subjected to SDS-PAGE. ab9879 and ab181602 (loading control to GAPDH) were both diluted at 1 μg/ml and 1/10000 respectively and incubated overnight at 4°C. Blots were developed withGoat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-USP9x antibody (ab19879)

Predicted band size: 292 kDa

false

• WB

Lab

Western blot - Anti-USP9x antibody (AB19879)

Lanes 1- 2 : Merged signal (red and green). Green - ab19879 observed at 290 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab19879 was shown to react with USP9x in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265665 (knockout cell lysate ab257790) was used. Wild-type HeLa and USP9X knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab19879 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-USP9x antibody (ab19879) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

USP9X knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human USP9X knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-usp9x-knockout-hela-cell-line-ab265665'>ab265665</a>)

Predicted band size: 292 kDa

Observed band size: 290 kDa

false

• WB

Unknown

Western blot - Anti-USP9x antibody (AB19879)

ab19879 detects full length USP9x protein as well as a number of USP9x fragments in WB on Caco2 Lysate :

289kDa Human protein : Q93008 USP9X (Full length protein)
105kDa Human protein : Q6P468 - USP9X protein (Fragment) Human protein
99.7kDa Human protein : Q59EZ5 - USP9X protein variant (Fragment).
53.9kDa Q86X58 - USP9X protein (Fragment).

All lanes:

Western blot - Anti-USP9x antibody (ab19879) at 1 µg/mL

Lane 1:

Caco-2 whole cell lysate (<a href='/en-us/products/unavailable/caco-2-whole-cell-lysate-ab3950'>ab3950</a>) at 20 µg

Lane 2:

Caco-2 whole cell lysate (<a href='/en-us/products/unavailable/caco-2-whole-cell-lysate-ab3950'>ab3950</a>) at 20 µg with Human USP9x peptide (<a href='/en-us/products/unavailable/human-usp9x-peptide-ab20617'>ab20617</a>)

Secondary

All lanes:

Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/5000 dilution

Predicted band size: 292 kDa

Observed band size: 100 kDa,105 kDa,290 kDa,50-65 kDa

true