Semi-quantitative western blotting was used to analyze the abundance of PNPLA3 in adipose, liver tissue, and primary hepatocytes. Detailed sample preparation and Western methods are provided in the Supplemental Methods . Due to apparent differences in relative PNPLA3 abundance between tissues, the primary PNPLA3 antibody (ab81874; Abcam, Cambridge, MA) was diluted to 1:250 for adipose tissue and 1:500 liver tissue and primary bovine hepatocyte samples for a 1 h room temperature incubation. After primary incubation, blots were washed and the secondary antibody (diluted 1:5000 for all samples types; ab97080, Abcam) and HRP conjugate (161-0381; Bio-Rad Laboratories, Richmond, CA) were applied for 1 h at room temperature. Blot images for total lane protein and the PNPLA3 band were captured on the ChemiDoc XRS + Imager using Image Lab 5.0 software (Bio-Rad Laboratories, Richmond, CA). The abundance of PNPLA3 protein was normalized to total lane protein within Image Lab 5.0 (Bio-Rad Laboratories, Richmond, CA) for statistical evaluation. Hepatocyte blots were also probed for ATGL abundance with the procedure detailed above, except the primary antibody (ab99532; Abcam) diluted to 1:3000 was incubated overnight at 4 °C and a different secondary antibody (ab97051, Abcam) diluted 1:5000 was used (Supplemental Methods ).
Image lab 5
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom, France, Australia
Image Lab 5.0 software is a powerful image analysis tool developed by Bio-Rad. It provides a comprehensive suite of tools for analyzing and quantifying images from various life science applications, including Western blots, gels, and blots. The software offers a user-friendly interface and advanced features for data processing and visualization.
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Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (66 mentions)
Chemidoc touch imaging system, by Bio-Rad (34 mentions)
Clarity western ecl substrate, by Bio-Rad (24 mentions)
Chemidoc mp, by Bio-Rad (24 mentions)
Pvdf membrane, by Merck Group (24 mentions)
Chemidoc xrs system, by Bio-Rad (24 mentions)
Chemidoc mp system, by Bio-Rad (21 mentions)
Chemidoc, by Bio-Rad (20 mentions)
Chemidoc xrs imaging system, by Bio-Rad (19 mentions)
Chemidoc xr, by Bio-Rad (19 mentions)
Pvdf membrane, by Bio-Rad (17 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
Chemidoc imaging system, by Bio-Rad (12 mentions)
Polyvinylidene fluoride membrane, by Merck Group (11 mentions)
Ripa buffer, by Merck Group (11 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
β actin, by Cell Signaling Technology (8 mentions)
β actin, by Merck Group (8 mentions)
526 protocols using image lab 5
1
Quantifying Adipose and Liver PNPLA3 Protein
2
Quantification of Chloroplast SOD and MDH
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The SOD isoforms in the isolated chloroplasts were identified in 12% native acrylamide gel, as described by Sehmer et al. (1998) (link). The exposure of bright areas was based on the inhibition of nitroblue tetrazolium (NBT) reduction by SOD activity and was quantified through the densitometry of bands using Image-Lab 5.2 software (Bio-Rad), after the correction for background.
The activity of NAD+-MDH was detected after the separation of foliar protein extracts in 8% native acrylamide gel, as described by Beeler et al. (2014) (link). The enzyme activity was developed with 1 mM malate, 0.5 mM NAD+, 0.4 mM NBT, 27 µM phenazine methosulfate, and 15 mM MgCl2 in 100 mM Tris-HCl with pH 8.0. The exposure of purple formazan bands was quantified through the densitometry of bands using Image-Lab 5.2 software (Bio-Rad), after the correction for background. The activity of NADP+-MDH was spectrophotometrically assayed as the NADPH-dependent reduction of OAA (Keryer et al., 2004 (link)). The maximal enzyme activity was measured after the reductive activation of extracts with 10 mM DTT for 30 min at RT.
Protein concentration was measured according to the methods of Bradford (1976) (link) using bovine serum albumin as the standard.
The activity of NAD+-MDH was detected after the separation of foliar protein extracts in 8% native acrylamide gel, as described by Beeler et al. (2014) (link). The enzyme activity was developed with 1 mM malate, 0.5 mM NAD+, 0.4 mM NBT, 27 µM phenazine methosulfate, and 15 mM MgCl2 in 100 mM Tris-HCl with pH 8.0. The exposure of purple formazan bands was quantified through the densitometry of bands using Image-Lab 5.2 software (Bio-Rad), after the correction for background. The activity of NADP+-MDH was spectrophotometrically assayed as the NADPH-dependent reduction of OAA (Keryer et al., 2004 (link)). The maximal enzyme activity was measured after the reductive activation of extracts with 10 mM DTT for 30 min at RT.
Protein concentration was measured according to the methods of Bradford (1976) (link) using bovine serum albumin as the standard.
3
Electrophoretic Analysis of PDGFRβ Expression
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Gel electrophoresis for PDGFRβ was performed using 10 µL of amplified PCR samples as described above. Gels were 2% agar made from Bio-Rad Certified Molecular Biology Agarose and 1× Tris base, acetic acid, and EDTA buffer. SYBR® Safe DNA Gel Stain (Invitrogen) was used to probe for bands. The expected size of target bands was confirmed with Thermo Fisher Scientific GeneRuler™ Express DNA Ladder. Equal loading was confirmed with β-actin. Immunoblots were developed using ChemiDoc™ MP Imaging System and Image Lab™ 5.0 (Bio-Rad).
4
Western Blot Protein Quantification
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Once cells were 80% confluent, they were lysed with appropriate buffer. Protein concentrations were determined using the Pierce BCA Protein Assay kit (NCI1059CH, Thermo, Waltham, MA, USA) and then adjusted to the same level. Proteins were denatured by heating at 100 °C for 5 min, subjected to SDS-PAGE electrophoresis, and transferred to membranes. For immunoblotting, the membranes were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies for 1 h at room temperature. Enhanced chemiluminescent substrate was added for gel imaging or film exposure. Images were collected using a Chemi DOC XRS + Imaging System (BioRad, Hercules, CA, USA). The gray scale value of protein bands was scanned using Image Lab5.0 (Biorad, Hercules, CA, USA).
5
Quantitative Immunoblotting for Fibrosis Markers
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Briefly, cell lysates (15 μg of protein per well) were separated by 10% SDS-PAGE gel, proteins were then transferred to nitrocellulose membrane, after which the blots were blocked with 5% skim milk in TBST buffer. The blots were washed three times in TBST buffer and then blots were probed using the mouse monoclonal αSMA (anti-αSMA, 1:1000; clone 1A4; ebioscience, Sandiego CA, USA), mouse monoclonal vimentin (anti-vimentin, 1:1000; Thermofisher Scientific, Waltham MA, USA). Loading control used was GAPDH (anti-GAPDH, 1:5000, Cell Signaling, Danvers MA, USA). Primary antibody incubated overnight at 4°C. Band intensities were detected, normalized and quantified with the Chemidoc and Image Lab 5.0 software (Bio-Rad Laboratories).
6
Western Blot Analysis of Cell Signaling
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The cells were collected and lysed in RIPA buffer (Cell Signaling Technology, USA). Homogenates of cells were clarified by centrifugation at 14,000 x g for 15 min at 4°C. The supernatants were collected, and the protein concentrations were measured by the BCA Protein Assay Kit (Sigma, USA). SDS-PAGE was performed on 40 μg of protein from each sample, gels were transferred to PVDF membranes (Millipore, USA) and incubated with the anti-GAPDH (Sigma, USA), anti-ALDH1A3 (Abcam, UK), anti-MMP2 (Abcam, UK), anti-snail and anti-slug (Cell Signaling Technology, USA). Followed by incubation with Peroxidase-Conjugated AffiniPure secondary antibody (ZSGB-BIO, China), the protein bands in the membranes were developed using Bio-Rad GelDoc XR System and analyzed by Image Lab 5.0 (Bio-Rad laboratories).
7
Collagen XVII and MMP9 Activation Assay
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The cells were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), supernatants were collected, and the protein concentrations were measured using a BCA Protein Assay Kit (Thermo, Waltham, CA, USA). SDS-PAGE was performed, subsequently transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) and incubated with anti-COL17A1(Immunogen: Synthetic peptide within Human COL17A1 aa 1300-1400) (Abcam, Cambridge, MA, USA), anti-MMP9 (Abcam, Cambridge, MA, USA), and anti-GAPDH (CWbiotech, Beijing, China). Rac Activation Assay Kit (NewEast Biosciences, USA) was used to detect the active form of RAC1 by pull-down assay. The blots were developed using the Bio-Rad GelDoc XR System and analyzed using Image Lab 5.0 (Bio-Rad Laboratories, Hercules, CA, USA).
8
Proteomic Analysis of Scorpion Venom Effects
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The experiments were performed on the W3 Western Workflow complete system (Bio Rad, USA). Equal amounts of protein isolated from breast and colorectal cell lines after treatment with scorpion venom (80 µg/mL) were mixed with 4× Laemmli’s buffer.24 (link) The samples were boiled at 97°C for 7 minutes and then separated on 4% to 12% Sodium Dodecyl Sulfate (SDS) gels. Proteins were transferred onto the pretreated Polyvinylidene Fluoride (PVDF) membranes. After blocking the membranes in 3% BSA for an hour, they were probed with 1:1000 diluted rabbit monoclonal p-Erk1/2, p-STAT3, RhoC, and GAPDH (Ab cam, UK) and BCLXL, BID, and p53 (Sigma Chemical Co, USA). After overnight incubation at 4°C, membranes were washed and blotted for 1 hour with horse radish peroxidase–conjugated antirabbit secondary antibodies (1:2500). Next, protein bands were visualized in a Chemi doc imaging system using ECL detection reagents; band density was calculated using image Lab 5.0 (Bio Rad, USA) and normalized against corresponding total proteins or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The normalized values are depicted in the form of bar graphs.
9
Protein Expression and Phosphorylation Analysis
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Muscle and liver biopsy specimens were homogenized in RIPA buffer containing a mixture of protease inhibitors. Homogenates were cleared by centrifugation (13.000 rpm; 30 min, 4 °C). Protein content was determined using Bradford Protein Assay. Protein lysates (30 μg) were separated on 10% SDS-PAGE and transferred on PVDF membrane. Membranes were probed overnight with Plin2, phospho-AktSer473, phospho-GSK3αβ Ser21/9, and Tubulin. Membranes were stripped for 30 min at 56 °C and re-probed overnight with total Akt or total GSK3αβ. Detection and analysis were performed respectively with Chemidoc XRS Image system and Image Lab 5.0 software (Bio-Rad Laboratories, Hercules, CA). Plin2 was normalized by Tubulin, while phospho-AktSer473 and phospho-GSK3αβ Ser21/9 resulted from the ratio of phosphorylated to total protein.
10
Profiling Serine Hydrolase Activities
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Activity-based protein profiling (ABPP) was performed by incubating
lysates with an inhibitor, then with the fluorophosphonate probe,
followed by separation by SDS-PAGE and visualization. Inhibitor (in
1 μL of DMSO) was added to a tube containing either brain or
liver tissue homogenate (2 mg/mL, 50 μL) and incubated for 20
min at 30 °C. ActivX TAMRA-FP serine hydrolase probe (1 μL,
Thermo Scientific, Waltham, MA) ([TAMRA-FP]final = 2 μM)
was added, and the mixture was incubated another 20 min at 30 °C.
The reaction was quenched with 4× loading buffer, boiled at 90
°C for 5 min, and loaded on a Bolt 4–12% Bis–Tris
SDS-PAGE gel (Thermo Scientific, Waltham, MA). The proteins were separated
at 200 V for 35 min run in MOPS SDS buffer (Thermo Scientific, Waltham,
MA). The bands were visualized using Bio-Rad ChemiDoc MP (Bio-Rad,
Hercules, CA) using a Cy3 filter and analyzed using ImageLab 5.0 (Bio-Rad,
Hercules, CA).
lysates with an inhibitor, then with the fluorophosphonate probe,
followed by separation by SDS-PAGE and visualization. Inhibitor (in
1 μL of DMSO) was added to a tube containing either brain or
liver tissue homogenate (2 mg/mL, 50 μL) and incubated for 20
min at 30 °C. ActivX TAMRA-FP serine hydrolase probe (1 μL,
Thermo Scientific, Waltham, MA) ([TAMRA-FP]final = 2 μM)
was added, and the mixture was incubated another 20 min at 30 °C.
The reaction was quenched with 4× loading buffer, boiled at 90
°C for 5 min, and loaded on a Bolt 4–12% Bis–Tris
SDS-PAGE gel (Thermo Scientific, Waltham, MA). The proteins were separated
at 200 V for 35 min run in MOPS SDS buffer (Thermo Scientific, Waltham,
MA). The bands were visualized using Bio-Rad ChemiDoc MP (Bio-Rad,
Hercules, CA) using a Cy3 filter and analyzed using ImageLab 5.0 (Bio-Rad,
Hercules, CA).
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