- 433900 - Invitrogen Antibodies RXRA antibody

Li Y, Zhu Y, Feng S, Ishida Y, Chiu TP, Saito T, Wang S, Ann DK, Ou JJ
Cell reports 2022 Jan 25;38(4):110284

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence of RXR-alpha was performed using HCT116 wild-type and RXR-alpha KO cells that were transfected with a green or a far-red fluorescent dye, respectively. Post-transfection, WT and KO cells were mixed and plated to a 1:1 ratio on coverslips as a mosaic and incubated for 24 hrs. Cells were fixed in 4% PFA (in PBS) or methanol for 15 min; cells were permeabilized with 0.1% Triton X-100 for 10 min at RT and blocked with PBS with 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 min. Cells were stained with the RXR-alpha monoclonal antibody (Product # 433900) at a 1:1,000 dilution overnight at 4°C. Secondary antibody incubation was performed using 1 µg/mL of Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 antibody (Product # A21424) together with DAPI for 1 hr. Imaging was performed with a 40X oil objective and analysis was performed using Image J. Cell image represents a single focal plane; WT and KO cells are outlined with a yellow (WT) or magenta (KO) dashed line. 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: Immunofluorescence analysis of RXRA was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with RXRA Monoclonal Antibody (Product # 433900) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of RXR-alpha was performed on HCT116 cell lysates. Antibody-bead conjugates were prepared by adding 1 µg of RXR-alpha monoclonal antibody (Product # 433900) with 30 µL of protein G-Sepharose beads and rocked overnight at 4°C. 1 mg 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 the same 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.

433900