Protein samples were isolated from colorectal cancer tissues using PARIS kit (Ambion). Total protein concentration was determined using Bradford protein reagent (Bio-Rad). Soluble proteins were loaded on precast TGX gels and were analyzed by immunoblotting with anti-TIGAR (dilution 1:1,000; Santa Cruz Biotechnology). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membrane was developed using C-Digit Blot Scanner (LI-COR, Hamburg, Germany). CRC cell line whole cell lysates were prepared as described (22 (link)). Soluble proteins were analyzed by immunoblotting with anti-TIGAR (Santa Cruz Biotechnology) and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membranes were developed using C-Digit Blot Scanner (LI-COR).
C digit blot scanner
Manufactured by LI COR
Sourced in United States, Germany, Japan, United Kingdom, China, Italy
The C-DiGit Blot Scanner is a compact, digital imaging system designed for Western blot analysis. It captures high-resolution images of chemiluminescent or fluorescent blots. The scanner features a charge-coupled device (CCD) camera and integrated software for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Image studio software, by LI COR (36 mentions)
Ripa buffer, by Thermo Fisher Scientific (30 mentions)
Westernsure premium chemiluminescent substrate, by LI COR (22 mentions)
Clarity western ecl substrate, by Bio-Rad (19 mentions)
Pvdf membrane, by Merck Group (18 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Pvdf membrane, by Bio-Rad (13 mentions)
Nitrocellulose membrane, by Bio-Rad (13 mentions)
β actin, by Cell Signaling Technology (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Trans blot turbo transfer system, by Bio-Rad (11 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
β actin, by Santa Cruz Biotechnology (10 mentions)
Anti β actin, by Merck Group (10 mentions)
β actin, by Merck Group (10 mentions)
Protease inhibitor, by Merck Group (9 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (9 mentions)
Ripa lysis buffer, by Merck Group (9 mentions)
Protease inhibitor cocktail, by Roche (9 mentions)
Nitrocellulose membrane, by GE Healthcare (9 mentions)
484 protocols using c digit blot scanner
1
TIGAR Protein Expression in Colorectal Cancer
2
Immunoblotting Technique for Autophagy Proteins
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The immunoblotting technique was performed as previously described [50 (link),72 (link)]. The antibodies used were Beclin 1 (#3738), ATG5 (#12994), LC3B (#2775), Sqstm1/p62 (#23214), and GAPDH (#2118) from CELL Signaling Technology (Danvers, MA, USA). All the primary antibodies were utilized at a dilution of 1:1000, and the secondary antibody (#7074s) from CELL Signaling Technology (Danvers, MA, USA) was used at a dilution between 1:10,000 and 1:20,000. GAPDH was used as a reference protein for the normalization of data. Ponceau S solution (#P7170) from SIGMA-ALDRICH (St. Louis, MO, USA) was used to verify total protein stain for immunoblotting normalization [73 (link)]. Images were acquired by the C-Digit Blot Scanner (LI-COR, Lincoln, Nebraska, USA) and quantified using the software Image Studio for C-Digit Blot Scanner.
3
Immunoblotting Analysis of Macrophages
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RAW 264.7 macrophages were treated for 24 h as previously described. Cell lysis buffer (120 mmol/L NaCl, 40 mmol/L Tris [pH 8], 0.1% NP40 with protease inhibitor cocktails (Sigma))
were used for the lysis of protein from cellular pellets. Proteins from cellular supernatants were separated on 8 to 10% polyacrylamide gel and transferred into a nitrocellulose membrane by using the trans-blot SD semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). Tris HCl buffered saline with Tween 20 (TBST) with 5% non-fat-milk were used for blocking the membrane. The primary antibodies 8-Oxoguanine glycosylase (OGG1), nuclear factor E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), SOD, CAT, heme oxygenase 1 (HO-1), TLR4, NF-кB, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IкBα), iNOS, TNF-α, IL-1β, IL-6, IL-10 and GADPH (1:500 dilutions) were used after all night incubation (4 °C). TBST were used for washing the membrane and incubated with secondary antibodies (1:80 000) for another 1 h. Immunolabeled proteins were recognized by using a chemiluminescence method (C-DiGit Blot Scanner, LICOR, Bad Homburg, Germany) and bands were quantified by using studio digits software 3.1 (C-DiGit Blot Scanner, LICOR, Bad Homburg, Germany).
were used for the lysis of protein from cellular pellets. Proteins from cellular supernatants were separated on 8 to 10% polyacrylamide gel and transferred into a nitrocellulose membrane by using the trans-blot SD semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). Tris HCl buffered saline with Tween 20 (TBST) with 5% non-fat-milk were used for blocking the membrane. The primary antibodies 8-Oxoguanine glycosylase (OGG1), nuclear factor E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), SOD, CAT, heme oxygenase 1 (HO-1), TLR4, NF-кB, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IкBα), iNOS, TNF-α, IL-1β, IL-6, IL-10 and GADPH (1:500 dilutions) were used after all night incubation (4 °C). TBST were used for washing the membrane and incubated with secondary antibodies (1:80 000) for another 1 h. Immunolabeled proteins were recognized by using a chemiluminescence method (C-DiGit Blot Scanner, LICOR, Bad Homburg, Germany) and bands were quantified by using studio digits software 3.1 (C-DiGit Blot Scanner, LICOR, Bad Homburg, Germany).
4
Western Blot Analysis of Oxidative Stress Markers
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Total cell lysate proteins were extracted and collected after the treatments. The protein concentration was detected by using the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). After the determination of total protein concentrations, the samples were denatured and loaded (40 μg/well) onto sodium dodecyl sulfate (SDS) polyacrylamide gels (Bio-Rad, Hercules, CA, USA) for electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) (Bio-Rad, Hercules, CA, USA) membrane by using electrophoretic transfer (Bio-Rad, Richmond, USA). Nonspecific reactivity was blocked for 1 h at room temperature with standard 1X tris-buffered saline—Tween 20 (TBST) (Sigma-Aldrich, St. Louis, MO, USA) buffer containing 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and 0.02% sodium azide. Primary antibodies of superoxide dismutase 1 (SOD-1), catalase, p53, Bad, Bax, Bcl-2, cytochrome c and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were probed and visualized on Li-COR C-digit Blot scanner (LI-COR Biosciences, Lincoln, Nebraska, USA) by using chemiluminescent reagent containing luminol.
5
Synaptic Protein Expression Analysis
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Synaptic proteins were assayed by western blot as described previously [2–4 (link)]. Briefly, aliquots of brain homogenates were mixed with equal volumes of Laemmi loading buffer and boiled prior to gel electrophoresis. Equal amounts of protein were loaded and separated using SDS-PAGE (4–20%; Bio-Rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were then rinsed in TBST buffer and incubated overnight in TBST solution containing the primary antibody of interest (goat anti-PSD-95 and rabbit anti-synapsin-1; Abcam, Cambridge, MA, USA). The next day, blots were incubated with the appropriate peroxidase-linked secondary antibody, followed by visualization of protein-antibody complexes using the enhanced chemiluminescence system (Millipore, Billerica, MA, USA), and digital images were developed using a Licor C-Digit blot scanner (LI-COR Biotechnology, Lincoln, NE, USA). Immunoreactive bands were compared densitometrically using the blot scanner’s software. Membranes were stripped off using a stripping buffer (Thermo Fisher Scientific, Rockford, IL, USA), and then incubated with β-tubulin antibody (mouse anti-βIII-tubulin; Sigma-Aldrich, St. Louis, MO, USA) used as the loading control.
6
Immunoblotting Protocol for Utrophin Detection
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophoshphate, 1 mM β-glycerophoshphate, 1 mM sodium orthovanadate) supplemented with complete protease inhibitor cocktail (Roche). Total protein was measured by Pierce BCA Protein assay kit (Thermo Fisher Scientific). 10 μg of total protein was resolved in 3%–8% Tris-Acetate protein gel (NuPAGE, Thermo Fisher Scientific) and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad). For immunoblotting, membranes were first blocked with 5% non-fat dry milk in TBS with 1% Tween20 for 1 h in room temperature. After blocking, blots were incubated with the following primary antibodies; mouse monoclonal anti-utrophin (1:100, Mancho3[8A4], developed by Prof. Glenn E. Morris; DSHB, IA, USA) and mouse anti-α-tubulin (1:5,000, T6199, Sigma-Aldrich) for overnight at 4°C. Next day, blots were washed; incubated with mouse immunoglobulin Gκ (IgGκ) binding protein (m-IgGκ BP) conjugated to horseradish peroxidase (HRP) (1:2,500, sc-516102, Santa Cruz Biotechnology); washed and developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged in LI-COR C-Digit Blot Scanner (LI-COR Biosciences-US).
7
Protein Expression Analysis Protocol
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Primary antibodies used were EGFR-t (C2C3, GeneTex), EGFR-p (S.684.2, Thermo), Src (36D10, Cell Signaling), Src-p (GTX24816, Genetex), STAT5 (9310, Cell Signaling), STAT5-p (9359, Cell Signaling), Sp1 (GTX110593, Genetex), Sp1-p (phosphor Thr739, Genetex), IGF1 (sc-9013, Santa Cruz Biotechnology), and β-actin (mouse monoclonal, CINVESTAV). Secondary antibodies used were goat anti-rabbit HRP (catalog number 62-6120, Invitrogen) or goat anti-mouse HRP (catalog number A9044, Sigma), WesternSure Chemiluminescent Western blotting reagent, and the Li-COR C-DiGit Blot Scanner (LI-COR Biosciences, Finland).
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted by lysis for 30 min on ice in RIPA buffer pH 7.6 (150 mM NaCL, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 7.6, 1% protease inhibitor cocktail (#P8340) and 1% phosphatase inhibitor (#P0044, both Sigma-Aldrich). Determination of protein concentrations and western blot analysis of whole-cell extracts was performed as described previously [88 (link)]. Primary antibodies were used against NRF2 (1:1000, ab62352; Abcam, Cambridge, UK), KEAP1 (sc-365626), p62/SQSTM1 (sc-28359), YAP (sc-398182; all 1:1000; Santa Cruz Biotechnology, Heidelberg, Germany), NF-κB p65 (#610868) and IKKα (#556532; both BD Bioscience, San Jose, CA, USA, 1:1000). Secondary antibodies were HRP-conjugated goat-anti-mouse or goat-anti-rabbit antibodies (1:5000, sc-2005 or sc-2004, Santa Cruz Biotechnology, Heidelberg, Germany). Anti-α-Tubulin (1:10000, B-512, Sigma-Aldrich) was used as loading control. Expression levels were visualised by WesternBright Quantum Kit (Biozym, Hessisch Oldendorf, Germany) or Super Signal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) on the Li-COR C-DiGit Blot Scanner using Image Studio Digits 4.0 software (LI-COR Biosciences, Lincoln, NE, USA).
9
Immunoblotting Protein Detection Protocol
10
Western Blot Analysis of CPT1A
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Cells were lysed with RIPA buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific) and sonicated for 30 seconds. Lysates were separated by SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and analyzed by immunoblotting. Rabbit anti-CPT1A (No. 12252) (Cell Signaling Technology) was used as a primary antibody. Immunoblotting for β-tubulin by mouse anti-β-tubulin antibody (Santa Cruz Biotechnology) and COX IV by rabbit anti-COX IV antibody (Cell Signaling) was used as a loading control for whole-cell lysates and mitochondrial lysates, respectively. Anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology and Thermo Fisher Scientific, respectively. Signal was detected using the ECL system with X-ray film development (Thermo Fisher Scientific and GE Healthcare Life Sciences) or a LI-COR C-Digit blot scanner (LI-COR) according to the manufacturer’s instructions.
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