- MA5-34809 - Invitrogen Antibodies RACK1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RACK1 in Lane 1: SH-SY-5Y, Lane 2: Rat small intestine. Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809), at a dilution of 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-RACK1 Recombinant Rabbit Monoclonal Antibody (JG40-26) (Product # MA5-34809) and a 35 kDa band corresponding to RACK1 was observed across across cell lines tested. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), THP-1 (Lane 2), Hep G2 (Lane 3), MCF7 (Lane 4), HeLa (Lane 5), PC-12 (Lane 6), Mouse Liver (Lane 7), Mouse Brain (Lane 8), Rat Liver (Lane 9) and Rat Brain (Lane 10) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RACK1 was achieved by transfecting HeLa with RACK1 specific siRNAs (Silencer® select Product # s20342, s20340). Western blot analysis (Fig. a) was performed using Whole cell extracts from the RACK1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RACK1 Recombinant Rabbit Monoclonal Antibody (JG40-26) (Product # MA5-34809, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 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 RACK1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of RACK1 was performed using 70% confluent log phase HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with RACK1 Recombinant Rabbit Monoclonal Antibody (JG40-26) (Product # MA5-34809) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing nucleus, cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of RACK1 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with RACK1 Recombinant Rabbit Monoclonal Antibody (JG40-26) (Product # MA5-34809) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing nucleus, cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of RACK1 in paraffin-embedded rat liver tissue. Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of RACK1 in paraffin-embedded rat cerebellum tissue. Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of RACK1 in paraffin-embedded mouse skin tissue. Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of RACK1 in paraffin-embedded human colon tissue. Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of RACK1 in SH-SY-5Y cells (purple) compared with an unlabelled control (cells without incubation with primary antibody; yellow). Samples were incubated with RACK1 monoclonal antibody (Product # MA5-34809) at a dilution of 1:100, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.

MA5-34809