Cell lysates, cell supernatants, purification fractions, rZNS1-His and gel filtration fractions were resolved on 7 % or 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), analysis by immunoblotting was performed using 0.2 μg/ml ZNS1 monoclonal antibody (mAbia labs, Argentina), 1:1000 glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich) or 0.1 μg/ml anti-His tag monoclonal antibody (mAbia labs, Argentina). Bound mAbs were recognized with a horseradish peroxidase (HRP) goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:2000 dilution, Alexa Fluor 680 goat anti-mouse IgG secondary antibody (Invitrogen) at a 1:20000 dilution, or with HRP rabbit anti-human IgG secondary antibody (Dako) at a 1:10000 dilution. The signal was visualized with enhanced chemiluminiscence reagent (GE Healthcare) and CL-XPosure Films (Thermo Scientific), or with an Odyssey Infrared Imager (Li-Cor). Densitometric analysis was performed using the NIH ImageJ software.
Enhanced chemiluminiscence reagent
Manufactured by GE Healthcare
Sourced in United States
Enhanced chemiluminescence reagent is a laboratory product designed to facilitate the detection and quantification of proteins in western blot analyses. The reagent produces a luminescent signal proportional to the amount of target protein present, allowing for sensitive and accurate measurements.
Automatically generated - may contain errors
Lab products found in correlation
Hrp goat anti mouse igg secondary antibody, by Merck Group (2 mentions)
Hybond ecl, by GE Healthcare (2 mentions)
Alexa fluor 680 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cl xposure film, by Thermo Fisher Scientific (2 mentions)
Hrp rabbit anti human igg secondary antibody, by Agilent Technologies (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Image studio lite software, by LI COR (1 mentions)
β casein, by Merck Group (1 mentions)
Rabbit anti actin antibody, by Merck Group (1 mentions)
Pdvf membrane, by GE Healthcare (1 mentions)
3 protocols using enhanced chemiluminiscence reagent
1
Immunoblotting Analysis of Protein Samples
2
Immunoblotting Analysis of Milk Proteins
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Food protein extracts, purified β-casein (Sigma-Aldrich) and fresh milk -UHT skim milk (milk), raw milk (a kind gift from Gabriela Rodriguez, INTI lacteos, Buenos Aires, Argentina), 10% skim milk powder (milk powder) and goat milk-, were resolved on 10% SDS-PAGE. After transfer to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), analysis by immunoblotting was performed using 0.02 μg/ml 1H3 or 0.2 μg/ml 6A12 purified monoclonal antibodies in blocking buffer (5% BSA in TBS). Bound antibodies were recognized with an HRP goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:6000 dilution in blocking buffer, or with Alexa Fluor 680 goat anti-mouse IgG secondary antibody (Invitrogen) at a 1:20000 dilution in blocking buffer. The signal was visualized with enhanced chemiluminiscence reagent (GE Healthcare) and CL-XPosure Films (Thermo Scientific), or with an Oddysey Infrared Imager (Li-Cor). Densitometric analysis was performed using the Li-Cor Image Studio Lite software.
3
Western Blot Protein Detection
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Protein extracts (15-25 μg per sample) from tissue samples were run in denaturing acrylamide gels, and then electrotransferred to PDVF membranes (GE Healthcare, Barcelona, Spain). Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Tween-20) containing 5% skimmed milk, and incubated with the primary K113 antibody. Detection was performed with an appropriate horseradish peroxidase-conjugated secondary antibody (EZBiolab, Carmel, IN, USA) and enhanced chemiluminiscence reagent (GE Healthcare). The K113 antibody was used at 1/5000 dilution; polyclonal rabbit anti-actin antibody (Sigma A2066, Madrid, Spain) at 1/1000; and secondary rabbit HRP-anti-Ig antibody (DakoCytomation, P0399, Madrid, Spain) at 1/10 000. Band pixel intensities were quantified using ImageJ (Wayne Rasband National Institutes of Health, Bethesda, MD, USA) and normalised to actin levels.
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