- 710388 - Invitrogen Antibodies NCAM1 antibody

Submitted references Purification of Pig Muscle Stem Cells Using Magnetic-Activated Cell Sorting (MACS) Based on the Expression of Cluster of Differentiation 29 (CD29).

Choi KH, Kim M, Yoon JW, Jeong J, Ryu M, Jo C, Lee CK
Food science of animal resources 2020 Sep;40(5):852-859

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NCAM was performed by loading 30 µg of HeLa (lane1), HEK-293 (lane2), SH-SY5Y (lane3), K562 (lane4) and U2OS (lane5) using Novex®NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5 % skim milk for 1 hour at room temperature. NCAM was detected at ~94, 140, 180 kDa using NCAM Recombinant Rabbit Polyclonal Antibody (Product # 710388) at 0.5 µg-1 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)
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Experimental details: Western blot was performed using Anti-CD56 Recombinant Polyclonal Antibody (3HCLC) (Product # 710388) and a 130 kDa band corresponding to CD56 was observed in NK-92. Membrane enriched cell extracts (12.5 µg lysate) of NK-92-Untreated (Lane 1), NK-92-Control (50°C, 3 hrs) (Lane 2) and NK-92-Treated (500 U PNGaseF; 50°C, 3 hr) (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright FL1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). CD56 migrates at ~130 kDa due to multiple glycosylations (Lane 1). To carry out deglycosylation of the protein, PNGase F Glycan Cleavage Kit was used (Product # A39245). Upon PNGaseF treatment, N-linked glycosylations are cleaved and deglycosylated CD56 can be seen at ~90-110 kDa (Lane 3). This mass shift can be attributed to deglycosylation as the control sample migrates at ~130 kDa (Lane 2).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CD56 Recombinant Polyclonal Antibody (3HCLC) (Product # 710388) and bands in the range of 130-240kDa corresponding to Neural cell adhesion molecule 1 were observed. Membrane enriched extracts (30 µg lysate) of SH-SY5Y (Lane 1), IMR-32 (Lane 2), NK-92 (Lane 3), SK-N-SH (Lane 4), MCF7 (Lane 5), SK-BR-3 (Lane 6) and T-47D (Lane 7) 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 (0.5 ug/ml) 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 Atto Ultimate Sensitivity Substrate (Product # A38556). Relative expression was observed between SH-SY5Y, IMR-32, NK-92, SK-N-SH and breast cancer cell lines such as MCF7, SK-BR-3 and T-47D as expected (DOI: 10.1038/s41598-019-45377-8).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NCAM in whole tissue extracts from rat brain using a NCAM Recombinant Rabbit Polyclonal Antibody (Product # 710388) at a dilution of 2 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~94kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NCAM in U2OS cells using a NCAM Recombinant Rabbit Polyclonal Antibody (Product # 710388) followed by detection using an Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing cytoplasmic localization of NCAM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of NCAM was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with NCAM Recombinant Rabbit Polyclonal Antibody (Product # 710388) at 1 µg-2 µg 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 with 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 and membrane localization of NCAM. Panel e shows no primary antibody. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Neural cell adhesion molecule 1 was performed using 70% confluent log phase IMR-32 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with CD56 Recombinant Polyclonal Antibody (3HCLC) (Product # 710388) at 1 ug/ml 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), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Plasma membrane localization. Panel e represents the merged image of MCF7 cells showing very low expression of CD56 as expected (DOI: 10.1038/s41598-019-45377-8). Panel f 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: Flow cytometry analysis of NCAM in K562 cells using a NCAM Recombinant Rabbit Polyclonal Antibody (Product # 710388). Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 Goat anti-Rabbit IgG (right peak) compared to an isotype control (left peak).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1. Purification of pig muscle stem cells by magnetic-activated cell sorting (MACS) using a CD29 antibody. Muscle stem cells isolated from the biceps femoris muscle of 14 d-old LYD pigs were sorted by MACS using a CD29 antibody. (A) The expression pattern of CD29 and CD56 in pig muscle stem cells as determined by immunostaining. Blue, green, and red fluorescence represent nuclei, CD29, and CD56, respectively. (B) The proportion of CD29- and CD56-positive cells in pig muscle stem cells as measured by immunostaining. (C) The proportion of CD29- and CD56-positive cells following MACS sorting was measured by immunostaining in cells from the various breeds (LYD, Berkshire, and Korean native pigs). Data are represented as mean+-SEM. The significance of the differences was determined between the unsorted and sorted groups. * p

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