- 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)
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Experimental details: Western blot of RXR-alpha was performed by loading 100 µg of WT (lane 1) and RXR-alpha CRISPR KO (lane 2) HCT116 cell lysates in RIPA buffer onto a 4-15% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. RXR-alpha was detected at approximately 50 kDa using an RXR-alpha monoclonal antibody (Product # 433900) at a dilution of 1:500 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. The peroxidase-conjugated secondary antibody (Product # 62-6520) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). 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)
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Experimental details: Knockdown of RXRA was achieved by transfecting MCF7 cells with RXRA specific siRNAs (Silencer® select Product # s12385, s12386). Western blot analysis (Fig. a) was performed using nuclear enriched extracts from the RXRA knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with RXRA Monoclonal Antibody (Product # 433900, 1 µg/mL dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to RXRA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on nuclear enriched extracts (30 µg lysate) of MCF7 (Lane 1), Caco-2 (Lane 2), A549 (Lane 3), Hep G2 (Lane 4), C2C12 (Lane 5), L6 (Lane 6), HeLa (Lane 7), A-431 (Lane 8) and NIH/3T3 (Lane 9). The blot was probed with Anti-RXRA Monoclonal Antibody (Product # 433900, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 54 kDa band corresponding to RXRA was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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)
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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)
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Experimental details: Immunohistochemistry analysis of RXR alpha in paraffin-embedded rat liver tissue. The sample was incubated in RXR alpha monoclonal antibody (Product # 433900) at a dilution of 3~20 µg/mL. Section was fixed with 10% formalin-fixed in immersion, activated by autoclave at 121°C for 15 minutes, and blocked with 3% H2O2 in methanol.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of RXR alpha in paraffin-embedded rat embryonic intestine. The sample was incubated in RXR alpha monoclonal antibody (Product # 433900) at a dilution of 3~20 μg/mL. Section was fixed with 10% formalin-fixed in immersion, activated by autoclave at 121°C for 15 minutes, and blocked with 3% H2O2 in methanol.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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