- PA5-18662 - Invitrogen Antibodies ITCH antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ITCH in U2OS cells using a ITCH monoclonal antibody (Product # PA5-18662) at 10 µg/mL for1hr. The cells were paraformaldehyde fixed and permeabilized with 0.15% Triton. Primary incubation was followed by Alexa Fluor 488 secondary antibody (2 µg/mL) showing nuclear staining. Actin filaments were stained with phalloidin (red). Negative control: Unimmunized goat IgG (10 µg/mL)followed by Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ITCH in A431 cells using a ITCH monoclonal antibody (Product # PA5-18662) at 10 µg/mL for1hr. The cells were paraformaldehyde fixed and permeabilized with 0.15% Triton. Primary incubation was followed by Alexa Fluor 488 secondary antibody (2 µg/mL) showing nuclear and vesicle staining. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 µg/mL)followed by Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of ITCH using ITCH Polyclonal Antibody (Product # PA5-18662) in paraformaldehyde fixed A431 cells, permeabilized with 0.15% Triton. Primary incubation 1hr (10 µg/mL) followed by Alexa Fluor 488 secondary antibody (2 µg/mL), showing nuclear and vesicle staining. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 µg/mL) followed by Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of ITCH using ITCH Polyclonal Antibody (Product # PA5-18662) in paraformaldehyde fixed U2OS cells, permeabilized with 0.15% Triton. Primary incubation 1hr (10 µg/mL) followed by Alexa Fluor 488 secondary antibody (2 µg/mL), showing nuclear staining. Actin filaments were stained with phalloidin (red). Negative control: Unimmunized goat IgG (10 µg/mL) followed by Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of ITCH was performed on HeLa cell lysates. Antibody-bead conjugates were prepared by adding 2 µg of ITCH polyclonal antibody (Product # PA5-18662) with 30 µL of protein G-Sepharose beads and rocked overnight at 4°C. 750 µg of lysate was incubated with an antibody-bead conjugate for 2 hours at 4°C. Following centrifugation and multiple washes, 10% starting material (SM), 10% unbound fraction (UB) and immunoprecipitated fraction (IP) were processed for immunoblot using a different antibody. Ponceau stained transfer of blot is shown. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of ITCH in HeLa cells using a polyclonal antibody (Product #PA5-18662). HeLa cells (blue line) were paraformaldehyde fixed and permeabilized with 0.5% Triton. The primary antibody was incubated for one hour (10 µg/mL) followed by an Alexa Fluor 488 secondary antibody (1 µg/mL). IgG control: Unimmunized goat IgG (black line) followed by an Alexa Fluor 488 secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of ITCH in HeLa cells using a polyclonal antibody (Product #PA5-18662). HeLa cells (blue line) were paraformaldehyde fixed and permeabilized with 0.5% Triton. The primary antibody was incubated for one hour (10 µg/mL) followed by an Alexa Fluor 488 secondary antibody (1 µg/mL). IgG control: Unimmunized goat IgG (black line) followed by an Alexa Fluor 488 secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of ITCH was performed on HeLa cell lysates. Antibody-bead conjugates were prepared by adding 2 µg of ITCH polyclonal antibody (Product # PA5-18662) with 30 µL of protein G-Sepharose beads and rocked overnight at 4°C. 750 µg of lysate was incubated with an antibody-bead conjugate for 2 hours at 4°C. Following centrifugation and multiple washes, 10% starting material (SM), 10% unbound fraction (UB) and immunoprecipitated fraction (IP) were processed for immunoblot using a different antibody. Ponceau stained transfer of blot is shown. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

PA5-18662