- MA1-016 - Invitrogen Antibodies LIN28A antibody

Submitted references SUMOylation modulates the LIN28A-let-7 signaling pathway in response to cellular stresses in cancer cells.

Dou J, Zhang H, Chen R, Shu Z, Yuan H, Zhao X, Wang Y, Huang J, Zhou A, Yu J
Molecular oncology 2020 Sep;14(9):2288-2312

Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation.

Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T
Stem cell reports 2015 Sep 8;5(3):448-59

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of LIN28 was performed by loading 75 µg of various whole cell lysates and 10 µl of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a LIN28 monoclonal antibody (Product # MA1-016) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-LIN28A Monoclonal Antibody (14E6-4E6) (Product # MA1-016) and a 32kDa band corresponding to LIN28A was observed in NTERA-2 and iPSC, but not in HeLa and SK-OV-3 which are reported to be negative. Modified whole cell extracts (1% SDS) (30ug lysate) of NTERA-2 (Lane 1), HeLa (Lane 2), SK-OV-3 (Lane 3) and iPSC (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of LIN28 in NCCIT and HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a LIN28 monoclonal antibody (Product # MA1-016) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502). F-Actin (red) was stained with DyLight-554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Lin28 (green) in HEL 11.4 induced IPS cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a Lin28 monoclonal antibody (Product # MA1-016) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a FITC-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Lin28 (green) in H9 embryonic stem cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a Lin28 monoclonal antibody (Product # MA1-016) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a FITC-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of LIN28A was performed using NTERA-2 and HeLa 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 1 hour at room temperature. The cells were labeled with LIN28A Monoclonal Antibody (14E6-4E6) (Product # MA1-016) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 (Product # A28175) at a dilution of 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 cytoplasmic expression of LIN28A in NTERA-2. Panel e represents HeLa cells, showing lesser expression of LIN28A. Panel f represents control NTERA-2 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 LIN28 showing staining in the cytoplasm of paraffin-embedded human seminoma (right) compared with 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 LIN28 monoclonal antibody (Product # MA1-016) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C 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 LIN28 showing staining in the nucleus and cytoplasm of paraffin-embedded mouse testis tissue (right) compared with 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 LIN28 monoclonal antibody (Product # MA1-016) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C 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: Flow cytometric analysis of Lin28 (blue histogram) on HEL 11.4 induced IPS cells. To generate single cells suspensions, colonies were treated with TrypLE cell dissociation enzyme for 5 minutes at 37°C. Cells were incubated with a Lin28 monoclonal antibody (Product # MA1-016) or mouse IgG (green histogram) at a dilution of 1:100 for 1 hour on ice, washed with PBS + 5% fetal calf serum (FACS buffer), and incubated with a FITC-conjugated secondary antibody at a dilution of 1:200 for 30 minutes on ice. Cells were washed with cold FACS buffer, resuspended in 500 µL of FACS buffer containing 10 µL of 4% paraformaldehyde, and analyzed on a flow cytometer.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of Lin28 (blue histogram) on H9 embryonic stem cells. To generate single cells suspensions, colonies were treated with TrypLE cell dissociation enzyme for 5 minutes at 37°C. Cells were incubated with a Lin28 monoclonal antibody (Product # MA1-016) or mouse IgG (green histogram) at a dilution of 1:100 for 1 hour on ice, washed with PBS + 5% fetal calf serum (FACS buffer), and incubated with a FITC-conjugated secondary antibody at a dilution of 1:200 for 30 minutes on ice. Cells were washed with cold FACS buffer, resuspended in 500 µL of FACS buffer containing 10 µL of 4% paraformaldehyde, and analyzed on a flow cytometer.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of LIN28 was performed using cross-linked chromatin from 1x10^6 HTC-IR rat hepatoma cells treated with insulin for 0, 10, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a LIN28 monoclonal antibody (Product # MA1-016). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 or exon-2-3 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the rat Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of LIN28 was performed using NCCIT whole cell lysate. Antigen-antibody complexes were formed by incubating 700 µg of lysate with 5 µg of an LIN28 monoclonal antibody (Product # MA1-016) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µl Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). NCCIT whole cell lysate (70 µg) was loaded as a positive control for detection. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a LIN28 monoclonal antibody (Product # MA1-016) at a dilution of 1:1000 overnight rotating at 4øC, washed in TBST, and probed with Clean-blot IP Detection Reagent (Product # 21230) at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

MA1-016

Antibody data