Clinical LF specimens (non-LFH group:LFH group = 2:2) were snap-frozen in liquid nitrogen and stored at −80°C for Western blotting. Total protein from each LF specimen was extracted in RIPA lysis buffer (Santa Cruz) and quantified using the BCA assay (Pierce). After denaturation, proteins specimens were separated using gel electrophoresis on 8%–12% SDS-PAGE, and then were transferred onto PVDF membranes (Roche Applied Science, Indianapolis, IN, USA). Using 5% nonfat dry milk for 2 hours at room temperature, The membranes were blocked in 5% nonfat dry milk for 2 h and then incubated overnight at 4°C with the following primary antibodies: Beclin1 (1:500; AF5128, Affinity), P62 (1:500; AF5384, Affinity), FN1 (1:500; AF5335, Affinity), TGFβ1 (1:500; AF1027, Affinity), NGF (1:500; AF5172, Affinity), HMOX1 (1:500; AF5393, Affinity), CAT (1:500; DF7545, Affinity), SIRT1 (1:500; TU365233, Abmart), and GAPDH (1:5000; AP0063, Bioworld). After, the membranes were incubated with goat anti-rabbit IgG (H+L) HRP secondary antibody (1:5000; RM3002, Rayantibody) for 2 h at room temperature. The proteins bands were detected using an enhanced chemiluminescence kit (KF005, Affinity), and chemiluminescence signals were quantified with Image Lab statistical software (Bio-Rad, Hercules, CA, USA).
Image lab statistical software
Manufactured by Bio-Rad
Sourced in United States
Image Lab is a statistical software designed for the analysis and visualization of gel and blot images. It provides tools for quantifying band intensity, detecting and analyzing bands, and generating publication-quality figures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Santa Cruz Biotechnology (2 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
P ampk, by Abcam (2 mentions)
Pvdf membrane, by Roche (1 mentions)
Ap0063, by Bioworld Technology (1 mentions)
Af5128, by Affinity Biosciences (1 mentions)
Af5384, by Affinity Biosciences (1 mentions)
Af5335, by Affinity Biosciences (1 mentions)
Af1027, by Affinity Biosciences (1 mentions)
Af5393, by Affinity Biosciences (1 mentions)
Kf005, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ab60171, by Abcam (1 mentions)
Ab170904, by Abcam (1 mentions)
Ab185210, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa kit, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using image lab statistical software
1
Investigating Protein Expression in Lacrimal Fluid
2
Immunoblot Analysis of Lens Proteins
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The immunoblot analysis was performed according to previous methods [6 (link)]. Briefly, the lenses were homogenized in lysis buffer (20 mM Tris-HCL (pH 7.4), 0.5 M EDTA (pH 8.0), 1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.32 mM sucrose, protease inhibitor cocktail). The lysates were sonicated for 30 min and centrifuged (16,000 ×g at 4 °C) for 15 min. The proteins were separated and transferred to the nitrocellulose membrane. Immunoblot analysis was performed using primary antibodies of m-calpain, Nrf-2, HO-1, PARP, and β-actin. The protein bands were visualized using a LAS3000 Luminescent image analyzer (Fuji Film, Tokyo, Japan). The protein expression levels were quantified with band density, with Image Lab statistical software (Bio-Rad, Richmond, CA).
3
Western Blot Analysis of Membrane Proteins
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Proteins (40 μg) in the membrane extracts or in the whole cell lysate were separated by 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat milk, the membrane was incubated with the primary antibodies against SLC7A11 (1:400, ab60171, Abcam, USA), P-gp (1:1000, ab170904, Abcam, USA), ATPase Na+/K+ (1:10000, ab185210, Abcam, USA) overnight at 4 °C, followed by incubation with the appropriate horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at 37 °C. The signals were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA) and were captured with a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA). The intensity of bands was quantified by Image Lab statistical software (Bio-Rad Laboratories, Hercules, CA, USA).
4
Western Blot Analysis of Protein Expression
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Extracted proteins from cells and tissues as directed by the radioimmunoprecipitation assay (RIPA) kit (Sigma-Aldrich, USA). Added 5–10 μL of the collected protein samples to the SDS-PAGE gel sample holes. Primary antibodies against the following targets were used: SIRT1 (Cat.No. 9475, CST), NAMPT (Cat.No. 236874, Abcam), IDO (Cat.No. 13268-1-AP, Sanying), AMPK (Cat.No. 32047, Abcam), p-AMPK (Cat.No. 131357, Abcam) and GAPDH (Cat.No. 8245, Abcam). The intensity of the bands on the western blots was evaluated by Image Lab statistical software (Bio-Rad, USA).
5
Immunoblot Protocol for Protein Analysis
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For immunoblot analysis, cells were lysed in loading buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 10% 2-mercaptethanol, and 0.01% bromophenol blue), separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes. Quantitative assessment of band intensity was performed using Image Lab statistical software (Bio-Rad) and ImageJ software (1.43r for Macintosh OS X; National Institute of Health). Uncropped versions of the most important blots are shown in Supplementary Fig. 16 .
6
Quantitative Protein Expression Analysis
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Western blotting was performed to detect protein levels in cells or tissues57 ,58 . Proteins were extracted in RIPA lysis buffer (Santa Cruz) and measured using a BCA protein assay kit (Thermo). After denaturation and SDS-PAGE electrophoresis, separated proteins were transferred to PVDF membranes and probed with primary antibodies and secondary HRP-conjugated IgG antibodies. Chemiluminescence signals were detected by Image Lab statistical software (Bio-Rad). Uncropped scans for western blot analysis were shown in Supplementary Figs. 14 –17 .
7
Western Blot Protein Analysis Protocol
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Western blot assay was performed as previously described20 (link). Tissue or cell extracts were prepared using a dounce homogenizer in cold RIPA buffer supplemented with cocktail. The homogenates were centrifuged at 12,000 rpm for 15 min, and the protein concentrations were determined using a protein assay from Thermo. The protein lysates were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The membrane was scanned using the Image Lab statistical software (Bio-Rad). Antibodies against PAR (Trevigen, 4335-MC-100), PARP1 (Trevigen, 4338-MC-50), p-γH2AX (Ser139) (Trevigen, 4418-APC-020), Gabarapl1 (Abcam, ab86497), ATG12 (Abcam, ab109491), P62 (Cell signaling Technology, 39749), LC3 (Cell Signaling Technology CST, 3868), p-P53 (Ser15) (Cell signaling technology, 9284), p-ATM (Ser1981) (Thermo Fisher MA1-2020),FoxO3a (Cell signaling technology, 12829), pFoxO3a (Cell signaling technology, 9466), β-actin (Cell signaling technology, 3700), PGC1α (Abcam, ab54481), TFAM (Abcam, ab131607), NRF1 (Abcam, ab175932), Lamin B1 (Cell signaling technology,13435), α-tubulin (Cell signaling technology,3873) were used as primary antibodies.
8
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from cells and tissues as directed by the radioimmunoprecipitation assay kit (Sigma-Aldrich). An appropriate amount of concentrated sodium dodecyl sulfate polyacrylamide gel electrophoresis protein loading buffer was added to the collected protein samples, and then 5-10 μL of the sample was loaded in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel sample holes. Low voltage constant pressure electrophoresis for the upper gel and high voltage constant voltage electrophoresis were applied, when bromophenol blue entered the lower gel. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes. The following primary antibodies were used: Sirt1 (Cat. No. 9475, Cell Signaling Technology, Danvers, MA, United States), peroxisome proliferator-activated receptor alpha (PPARα, Cat. No. 23398R, Bioss, Woburn, MA, United States), HIF-1α (Cat. No. 20398R, Bioss), AMP-activated protein kinase (AMPK, Cat. No. 32047, Abcam, Cambridge, United Kingdom), p-AMPK (Cat. No. 131357, Abcam), Bnip3 (Cat. No. 109414, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (Cat. No. 8245, Abcam). Image Lab statistical software (Bio-Rad, Hercules, CA, United States) was used to evaluate band intensities on Western blots.
9
Western Blotting of AMPK, mTOR, and AKT
10
Western Blot Analysis of Caspase-3
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The resected lenses were homogenized in lysis buffer (20 mM Tris-HCL (pH 7.4), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 M EDTA (pH 8.0), 0.32 mM sucrose, protease inhibitor cocktail, 1 mM NaF, 1 mM Na3VO4). Total tissue lysates were dispersed ultrasonically for 30 min and centrifuged 16,000 × g at 4 °C for 15 min. The proteins were separated with 12.5% PAGE (PAGE) and then transferred to a nitrocellulose membrane. The primary antibodies of caspase-3 (Cell Signaling Technology, Danvers, MA) were used at a dilution of 1:1,000 and incubated at 4 °C overnight. After incubation, the membranes were incubated with horseradish peroxidase (HRP)–labeled anti-rabbit second antibodies for 2 h at 1:5,000 in blocking solution. Expression levels were quantified by band density, followed by normalization to the beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) control. The bound antibody was visualized using a LAS3000 Luminescent image analyzer (Fuji Film, Tokyo, Japan). Expression levels of band intensity were quantified by image Lab statistical software (Bio-Rad, Richmond, CA).
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