Cells were harvested after treatment and homogenized in RIPA lysis buffer (Beyotime, P0013B) supplemented with complete EDTA-free cocktail protease inhibitor (Roche). The extracted protein samples were resoluted by 10% SDS-PAGE gels and then transferred onto nitrocellulose (NC) membranes (Millipore, Bedford, MA). The NC membranes were blocked with 5% nonfat dry milk in TBST for 1 h, and then probed with primary antibodies overnight at 4°C. At the second day, the NC membranes were washed by TBST for 3 times, and further probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The protein bands were acquired by incubating with the enhanced chemiluminescence (ECL) (Amersham Biosciences, Amersham, UK). The antibodies used in this study were purchased from the following sources: anti-Bax antibody (Novus Biologicals, NBP1-28566); anti-Bcl-2 antibody (Santa Cruz, sc-7382); anti-cleaved caspase-3 (Asp175) (Cell Signaling Technology, 9661); anti-ALDH1A1 antibody (Abcam, EP1933Y); anti-β-Actin (Abcam, ab14128); anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074); anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, 7076).
Anti aldh1a1 antibody
Manufactured by Abcam
Sourced in United States
The Anti-ALDH1A1 antibody is a research-use-only tool that specifically binds to the ALDH1A1 protein. ALDH1A1 is an enzyme involved in the oxidation of retinol to retinoic acid. The antibody can be used to detect and quantify ALDH1A1 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (1 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Anti bcl 2 antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Plvx mcherry n1 plasmid, by Takara Bio (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Alexa 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti mouse cd45 antibody, by BioLegend (1 mentions)
Alexa 647 conjugated anti human cd31 antibody, by BioLegend (1 mentions)
Pe conjugated anti human cd45 antibody, by BioLegend (1 mentions)
Anti cd13 antibody, by Abcam (1 mentions)
5 protocols using anti aldh1a1 antibody
1
Western Blot Analysis of Apoptosis Markers
2
Immunofluorescence Analysis of LOX and ALDH1A1 Expression
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The cells were seeded into 24-wells chamber glass slides and grown to a near-confluent state. Immunofluorescence analysis was performed according the standard procedure. Briefly, cells were washed two times with PBS, fixed and permeabilized with ice-cold acetone/methanol (1:1) for 10 min, rinsed with PBS and blocked in 3% BSA for 30 min at room temperature. After blocking, cells were incubated in primary antibody solution against LOX (rabbit polyclonal anti-LOX antibody, 1:100, Proteintech, Manchester, UK) and ALDH1A1 (rabbit monoclonal anti-ALDH1A1 antibody, 1:100, Abcam, Cambridge, UK) for 2 h at room temperature. Afterwards, cells were washed three times with PBS and incubated in Alexa Fluor®488 secondary antibody solution (Donkey Anti-Rabbit IgG, Jackson ImmunoResearch Laboratories, Cambridgeshire, UK) for 1 h at room temperature. Slides were washed with PBS and mounted in DAPI mounting medium. The expression analysis and pictures were taken under fluorescence microscope (Zeiss Axio-Imager.Z1).
3
Overexpression and Mutagenesis of ALDH Genes
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Full-length human ALDH1A1 and ALDH3A1 were amplified and cloned into the pLVX-mCherry-N1 plasmid (Clontech) under the control of the cytomegalovirus (CMV) promoter. Point mutations were generated using a Quickchange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. LbNOX gene was synthesized according to the provided sequence [18 (link)] and cloned into Lenti-blasticidin plasmid (Addgene). The following antibodies and reagents were used: anti-ALDH1A1 antibody (Abcam); anti-ALDH3A1 (Santa Cruz); anti-GAPDH antibody (Cell Signaling Technology); anti-mCherry antibody, which was a gift from Dr. Xiaocheng Wang's lab at NIBS.
4
Multicolor Flow Cytometry of Cells
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The following antibodies were used: purified rat anti‐mouse CD31 antibody (BD Pharmingen), Alexa 647‐conjugated anti‐mouse CD31 antibody, APC‐conjugated anti‐mouse CD45 antibody, purified mouse anti‐human CD31 antibody, Alexa 647‐conjugated anti‐human CD31 antibody, PE‐conjugated anti‐human CD45 antibody (BioLegend, San Diego, CA, USA), anti‐ALDH1A1 antibody (Abcam, Cambridge, UK), Alexa 594‐conjugated anti‐rabbit IgG, and Alexa Fluor 568‐conjugated anti‐rat IgG antibody (Invitrogen, Tokyo, Japan).
5
Tumor Protein Expression Analysis
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The tumor tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. The concentrations of the extracted proteins were determined using the bicinchoninic acid (BCA) method. Equal amounts of total protein from each animal were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10-12% gels. After electrophoresis, the proteins were transferred onto the polyvinylidene fluoride (PVDF) membranes, which were incubated with the anti-CD90 antibody (Biorbyt Ltd., UK; 1:200 dilution), anti-notch1/2/3 antibodies (Beijing Biosynthesis Biotechnology Co., Ltd., China; 1:500 dilution), anti-CD13 antibody (Abcam, USA; 1:1000 dilution), anti-CD133 antibody (Abcam, USA; 1:2000 dilution), anti-EpCAM antibody (Proteintech Group, USA; 1:2000 dilution), and anti-ALDH1A1 antibody (Abcam, USA; 1:500 dilution) at 4°C overnight. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference in the western blot. The membranes were blocked with 5% skim milk for 2 hours and then incubated with the horseradish peroxidase-labeled anti-rabbit secondary antibody (1:3000 dilution; Biorbyt Ltd., UK). The proteins were visualized using a chemiluminescence method in a darkroom.
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