- PA1-096 - Invitrogen Antibodies LIN28A antibody

Submitted references Manipulations in HIWI level exerts influence on the proliferation of human non-small cell lung cancer cells.

Wang Y, Liu J, Wu G, Yang F
Experimental and therapeutic medicine 2016 May;11(5):1971-1976

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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 NCCIT, NTERA-2 and control HeLa whole cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for 1 hour. Membranes were probed with a rabbit polyclonal antibody recognizing LIN28 (Product # PA1-096) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of LIN28 was performed by loading 50 µg of F9, NIH3T3 and NRK 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 polyclonal antibody (Product # PA1-096) 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-rabbit HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-LIN28A Polyclonal Antibody (Product # PA1-096) 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-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 using anti-LIN28 polyclonal antibody (Product # PA1-096) shows specific expression in human embryonal carcinoma NTERA-2 cells (shown in green) but not in control 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 rabbit polyclonal antibody recognizing LIN28 (Product # PA1-096), at a dilution of 1:200 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Lin28 (red) 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 polyclonal antibody (Product # PA1-096) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a fluorescently-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 (red) 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 polyclonal antibody (Product # PA1-096) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a fluorescently-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)
Main image
Experimental details: Immunofluorescent analysis of LIN28 (green) in Hela cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 polyclonal antibody (Product # PA1-096) at a dilution of 1:100 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of LIN28 (green) in HepG2 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 polyclonal antibody (Product # PA1-096) at a dilution of 1:200 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of LIN28 (green) in murine cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 polyclonal antibody (Product # PA1-096) at a dilution of 1:200 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X 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 1% BSA for 1 hour at room temperature. The cells were labeled with LIN28A Polyclonal Antibody (Product # PA1-096) at 5 µg/mL 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 (Product # A27034) 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 was performed on human tonsil tissue. To expose target protein, antigen was retreived using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 Rabbit polyclonal antibody (Product # PA1-096) at a dilution of 1:50 overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on mouse heart tissue. To expose target protein, antigen was retreived using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 Rabbit polyclonal antibody (Product # PA1-096) at a dilution of 1:20 overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on human placenta tissue. To expose target protein, antigen was retreived using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LIN28 Rabbit polyclonal antibody (Product # PA1-096) at a dilution of 1:50 overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

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 polyclonal antibody (Product # PA1-096) or rabbit 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 fluorescently-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: Immunoprecipitation of LIN28 was performed on NCCIT cells. The antigen:antibody complex was formed by incubating 750 µg whole cell lysate with 3 µg of rabbit polyclonal antibody recognizing LIN28 (Product # PA1-096) overnight on a rocking platform at 4øC. The immune-complex was captured on 50 µL Protein A/G Plus Agarose (Product # 20423). Captured immune-complexes were washed and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). IP and lysate control samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. Membranes were probed with a rabbit polyclonal antibody recognizing LIN28 (Product # PA1-096) at a dilution of 1:1000 overnight at 4øC on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with Clean Blot IP Detection reagent (Product # 21230) at a dilution of 1:2000 for one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Pico (Product # 34078).

PA1-096

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