71-5300

antibody from Invitrogen Antibodies

Targeting: RAB11A YL8

Provider product page for 71-5300

Western blot

Antibody data

Antibody Data
Antigen structure
References [95]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [5]
Immunohistochemistry [3]
Flow cytometry [1]
Other assay [3]

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Product number: 71-5300 - Provider product page
Provider: Invitrogen Antibodies
Product name: RAB11A Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This polyclonal antibody reacts specifically with the human Rab11A protein. Cross-reactivity with other Rab family members has not been detected.
Concentration: 0.25 mg/mL

RAB11A protein structure - 71-5300 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HEK293 cell lysates using Rabbit anti-Rab11 Polyclonal Antibody (Product # 71-5300).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Ras-related protein Rab-11A was achieved by transfecting HeLa with Ras-related protein Rab-11A specific siRNAs (Silencer® select Product # S16703, S16704). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the Ras-related protein Rab-11A knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RAB11A Polyclonal Antibody (Product # 71-5300, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:2000 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 Ras-related protein Rab-11A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-RAB11A Polyclonal Antibody (Product # 71-5300) and a 25 kDa band corresponding to Ras-related protein Rab-11A was observed across cell lines and tissue extract tested. Membrane enriched extracts (30 µg lysate) of A549 (Lane 1), MCF7 (Lane 2), HeLa (Lane 3), A-431 (Lane 4), U-2 OS (Lane 5), C2C12 (Lane 6), PC-12 (Lane 7), COS-7 (Lane 8) and tissue extract (30 µg lysate) of Mouse Kidney (Lane 9) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of RAB11 was performed by loading 20 µg of Cos-7 (lane1), K562 (lane2), A431 (lane3) and SH-SY5Y (lane4) cell lysate using Novex®NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. RAB11 was detected at ~ 27 kDa using RAB11 Recombinant Rabbit Polyclonal Antibody (Product # 71-5300) at 1-2 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Rab11 (green) in 3T3 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes and blocked with 3% Blocker BSA (Product # 37525) in PBS for 30 minutes at room temperature. Cells were stained with or without Rab11 rabbit polyclonal antibody (Product # 71-5300), at a concentration of 5 µg/mL for 1 hour at room temperature, and then incubated with a Goat anti-Rabbit (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:1000 for 1 hour at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Ras-related protein Rab-11A was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with RAB11A Polyclonal Antibody (Product # 71-5300) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2,000), 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic (Golgi complex and cleavage furrow like pattern) localization. Panel e represents control cells with no primary antibody to assess background. 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 Ras-related protein Rab-11A was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with RAB11A Polyclonal Antibody (Product # 71-5300) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2,000), 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. 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 Ras-related protein Rab-11A was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with RAB11A Polyclonal Antibody (Product # 71-5300) at 1:100 in 0.1% BSA, incubated at 4 ° C overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. 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 Rab11 was done on 70% confluent log phase U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Rab11 Rabbit Polyclonal Antibody (Product # 71-5300) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of RAB11 showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a RAB11 polyclonal antibody (Product # 71-5300) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of RAB11 showing staining in the cytoplasm of paraffin-embedded human stomach tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a RAB11 polyclonal antibody (Product # 71-5300) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of RAB11 showing staining in the cytoplasm of paraffin-embedded mouse stomach tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a RAB11 polyclonal antibody (Product # 71-5300) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6. Downregulation of calsyntenin-1 by RNAi results in enhanced APP processing and increased secretion of Abeta. Cortical neurons were infected with rAAVs expressing a hairpin targeting calsyntenin-1 (shCst1) or a nonsense hairpin (shNS), together with eGFP. ( A ) Western blot of cell lysates. The signal indicated equal expression of the recombinant proteins. ( B ) Quantification of Western blots. Values indicate mean+-s.e.m. (*, P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 9 Characterization of purified ICVs. A549 cells were infected with the WSN virus and submitted to subcellular fractionation at 14 hpi. a Western-blot analysis of subcellular fractions of mock-infected (left panel) and IAV-infected (right panel) A549 cells, upon sucrose gradient ultracentrifugation. The same amount of total protein was analyzed for each fraction. TfR: transferrin receptor; PDI: protein disulfide isomerase; Cal: calreticulin; Clathrin HC: Clathrin Heavy Chain. For gel source data, Supplementary Fig. 17 . b TEM analysis of an aliquot of fraction 60-40% from IAV-infected cells, upon fixation and negative staining. Black arrows: ICVs. White arrows: vRNPs. Scale bar: large panel 200 nm, small pannel 100 nm

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1. Calsyntenin-1/APP vesicles contain BACE1, but no ADAM10. ( A ) Schematic representation of vesicle immunoisolation. Vesicle immunoisolations were performed with antibodies against cytosolic epitopes of vesicle-associated proteins and Dynabeads coated with protein A. Captured vesicles were isolated and washed. Vesicular proteins were eluted by lysis of the captured vesicles with a mild detergent. Note that the vesicular component bearing the antigen targeted for immunoisolation is not or only partially found in the eluate, because of its strong binding to the antibody/protein A/bead complex. Its complete elution required harsher conditions, e.g. boiling of the beads in SDS. Therefore, the antigen used for vesicle immunoisolation is not shown on the Western blots of the (mild) eluates. Examples of antigen elutions under harsh condition from the beads are shown in the panels labeled ""beads"". ( B ) Specificity tests of anti-APP, anti-ADAM10, and anti-BACE1 antibodies, performed by the addition of ~25 ug of recombinant antigen (GST-APPcyto, GST-ADAM10cyto, BACE1[485-501]). Saturation with recombinant antigen prevented vesicle capture. ( C ) Calsyntenin-1 organelles contained BACE1, but no ADAM10 (black box). ( D ) APP and syntaxin13 immunoisolates contain a low level of active ADAM10 and abundant BACE1. Neither BACE1 nor ADAM10 were found in Rab11 immunoisolates. ( E ) Rab5 immunoisolates contain a small amount of active ADAM10 (arrowhead) and abundant BACE1 (green box).