Proteins in total cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). PVDF was incubated with primary antibodies against eIF5a (ab32443; Abcam), CBL (ab52855; Abcam), or β‐actin (A5441; Sigma‐Aldrich LLC) overnight at 4°C. Subsequently, horseradish peroxidase‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG was applied at room temperature for 2 hours. Membranes were analyzed by a ChemiDoc XRS + image analyzer (Bio‐Rad).
Chemidoc xrs image analyzer
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc XRS+ image analyzer is a versatile laboratory equipment designed for high-resolution imaging and analysis of various samples, such as gels, blots, and microplates. It features a sensitive CCD camera, a range of illumination options, and powerful image processing software, enabling users to capture and analyze images with precision and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Western blocker solution, by Merck Group (3 mentions)
West pico plus hrp substrate, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Stain free 4 20 gels, by Bio-Rad (2 mentions)
0.45 micron pvdf membranes, by Bio-Rad (2 mentions)
Enhanced chemiluminescence assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg reagent, by Thermo Fisher Scientific (2 mentions)
Criterion tgx precast gel, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ab32443, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
27 protocols using chemidoc xrs image analyzer
1
Immunoblotting Analysis of Cellular Proteins
2
Western Blot Protein Analysis Protocol
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The cells were lysed in RIPA buffer with 1% PMSF. The proteins were separated on 10% SDS-polyacrylamide gels and were transferred to PVDF membranes (Millipore). The membranes were blocked in 5% fat-free milk in TBST and then incubated with primary antibodies overnight at 4 °C. After washed three times with TBST, membranes were incubated with secondary antibodies at room temperature for 1 hour and were washed three times again. Protein bands were visualized using an enhanced chemiluminescence reagent (GE Healthcare, UK) and quantified by densitometry using ChemiDoc XRS+ image analyzer (Bio-Rad, USA).
3
Western Blot Analysis of NF-κB Signaling
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Proteins were extracted from IPEC-J2 cells using RIPA lysis buffer (P0013B, Beyotime, China) and a Nuclear and Cytoplasmic Protein Extraction Kit (KGP1000, KeyGEN, China). Proteins were quantified using a BCA protein assay kit (KGP1100, KeyGEN). Proteins (20 μg/sample) were separated by SDS-PAGE (Tricine-SDS-PAGE Gel Kit, CW2384S, Cwbio, China), transferred to nitrocellulose membranes (88585, Pierce, Rockford, USA), and then hybridized with specific antibodies. The following antibodies were used: NF-κB p65 (#8242, Cell Signaling Technology, Danvers, MA, USA), phospho–NF-κB p65 (#3033, Cell Signaling Technology), IκBα (#4814, Cell Signaling Technology), phospho-IκBα (#2859, Cell Signaling Technology), Nrf2 (#12721, Cell Signaling Technology), anti–HO-1 (EP1391Y, Abcam, UK), lamin B1 (ab16048, Abcam, UK), and β-actin (CW0096, Cwbio). Total protein and cytoplasmic protein levels were normalized using β-actin expression to correct for differences in protein loading. Nuclear protein blots were normalized using lamin A/C to correct for differences in protein loading. Blots were visualized using an ECL detection system, and proteins were quantified using a ChemiDoc XRS+ image analyzer (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Affibody Constructs
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The samples and controls were loaded and separated with reducing SDS-PAGE. Stain free 4–20% gels were used (Bio-rad). The proteins were transferred to 0.45-micron PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad). The blot was blocked using the Western blocker solution (Sigma Aldrich) and incubated in with either anti-Z-domain (2.87 mg/mL) (1:1000) or anti-ABD (1 mg/mL) (3:1000) antibodies, both obtained from Affibody AB followed by incubation with either anti-mouse antibody (1:5000) for anti-Z-domain or anti-rabbit (1:5000) antibody for anti-ABD antibodies, respectively. Both secondary antibodies were HRP-conjugated and visualized by using West Pico Plus HRP substrate (Thermo Fischer) and measured with a ChemidoC XRS image analyzer (Bio-Rad). As positive controls, standards of purified ZHER3_1-ABD (1.97 mg/mL), ZHER3_1-ZHER3_1-ABD (0.77 mg/mL) and ZHER3_1-ABD-ZHER3_1 (1.34 mg/mL) provided by Affibody AB were used with a concentration of 0.01 g/L or stated otherwise.
5
Western Blotting Analysis of Skin Tissue and Cells
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Protein samples were prepared from the skin tissues, and the HaCaT cells were cultured with 1% Triton-X radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail. The protein concentrations of the lysed cells and homogenized skin tissue samples were determined using the bicinchoninic acid assay. Equal amounts of protein were loaded onto 8%~10% sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1 hour. Subsequently, the membranes were incubated overnight with primary antibodies at 4℃; subsequently, they were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein expression was detected using the EzWestLumi Plus system (ATTO, Tokyo, Japan), and images were captured by exposing the membranes to a ChemiDoc™ XRS image analyzer (Bio-Rad, Hercules, CA, USA).
6
Western Blot Protein Detection Protocol
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Western blot was conducted following standard procedures. Briefly, the cells were lysed with 1% Triton-X radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail. The remaining cell debris was pulled out by centrifugation (13,200 rpm, 15 minutes), and the protein concentration was determined by the bicinchoninic acid assay method. Identical amounts of protein were loaded on 8%~15% sodium dodecyl sulfate-polyacrylamide gels and separated by electrophoresis. After electrophoresis, the proteins were transferred on nitrocellulose membranes, which were blocked with 5% skim milk for 1 hour. Subsequently, the membranes were incubated with primary antibodies (1:1,000 dilution) overnight at 4℃, and with horseradish peroxidase-conjugated secondary antibodies (1:2,000 dilution) for 1 hour at room temperature. Proteins were detected using the EzWestLumi plus system (ATTO, Tokyo, Japan), and images obtained using a ChemiDoc™ XRS image analyzer (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of Adipogenic Markers
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Cells were washed and harvested with ice-cold DPBS. Lysates were prepared using radioimmunoprecipitation cell lysis buffer containing 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxylcholic acid, 10% NP-40 and 10 mM EDTA. Protein concentration was measured using a Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific Inc.). Cell lysates (30 µg) were separated by 4–20% Criterion™ TGX™ precast gel (Bio-Rad Laboratories, Inc.) electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked with 5% skimmed milk and incubated overnight at 4°C with C/EBPα, PPARγ and β-actin primary antibodies at a dilution of 1:1,000. After 1 h incubation at room temperature with HRP-conjugated anti-mouse for or anti-rabbit secondary antibodies (1:3,000), protein bands were detected with an Enhanced Chemiluminescence assay kit (Thermo Fisher Scientific, Inc.). Images were captured using a ChemiDoc™ XRS+ image analyzer (Bio-Rad Laboratories, Inc.). Relative density of C/EBPα and PPARγ were normalized to β-actin and quantified using Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc.).
8
Immunoblotting of RadA Protein
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Cells were cultivated as described previously in 30 mL. Cells from strains in the stationary or exponential growth phase were harvested and resuspended in PBS buffer complemented with protease inhibitors (Roche, #05056489001). The cells were disrupted after 5 min in ultrasonic bath, and cellular extracts were collected from the supernatant after centrifugation at 10,000 × g for 60 min. The concentrations of total protein were measured by Bradford protein assay (Bio-Rad, #500-0205).
Cell extracts were separated by denaturing electrophoresis (Bio-Rad, #4568094) and transferred onto a PVDF membrane (Bio-Rad, #1704156) during 3 min at 25 V with the Trans-blot turbo transfer system (Bio-Rad, #1704150). The blots were blocked with 5% milk in PBS-T for 60 min and incubated with primary antibody 1:5,000 for 60 min and secondary antibody 1:10,000 for 60 min. Anti-RadA antibodies (gift from Ishino’s lab) were prepared by immunizing rabbits with the recombinant P. furiosus RadA. Anti-rabbit IgG HRP (GE Healthcare, NA934V) was used as the secondary antibody. Proteins were visualized with an enhanced chemiluminescence detection system (Thermo Fisher, 34076) and a Chemidoc-XRS image analyzer (Bio-Rad).
Cell extracts were separated by denaturing electrophoresis (Bio-Rad, #4568094) and transferred onto a PVDF membrane (Bio-Rad, #1704156) during 3 min at 25 V with the Trans-blot turbo transfer system (Bio-Rad, #1704150). The blots were blocked with 5% milk in PBS-T for 60 min and incubated with primary antibody 1:5,000 for 60 min and secondary antibody 1:10,000 for 60 min. Anti-RadA antibodies (gift from Ishino’s lab) were prepared by immunizing rabbits with the recombinant P. furiosus RadA. Anti-rabbit IgG HRP (GE Healthcare, NA934V) was used as the secondary antibody. Proteins were visualized with an enhanced chemiluminescence detection system (Thermo Fisher, 34076) and a Chemidoc-XRS image analyzer (Bio-Rad).
9
Western Blot Analysis of EMT Markers
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Western blotting was performed using an enhanced chemiluminescence kit (Merck Millipore), as described previously.15 The following antibodies were used: myc (PL14) and β‐actin (M177‐3) from MBL (Nagoya, Japan); E‐cadherin (#3195), N‐cadherin (#13116), Smad2/3 (#8685), phospho‐Smad2 (#3108), phospho‐Smad3 (#9520), Snail (#3879), Slug (#9585), HMGA2 (#5269) from Cell Signaling Technology (Beverly, MA); TEAD4 (ab58310) from Abcam (Cambridge, UK); GFP (mFX75) from Fujifilm Wako (Tokyo, Japan); TetR (TET01) from MoBiTech (Cambridge, MA, USA); and TWIST (25465–1‐AP) from Proteintech (Rosemount, IL, USA). Horseradish peroxidase (HRP)‐F(ab’)2 secondary antibodies were purchased from GE Healthcare (Waukesha, WI, USA). Protein bands were analysed using a ChemiDoc XRS+image analyzer (Bio‐Rad, Hercules, CA, USA). The intensity of bands was measured by Quantity One software (Bio‐Rad). Quantitative ratios of E‐cadherin, N‐cadherin, Snail, Slug, HMGA2 or TEAD4 to actin and EGFP to TetR, which were calculated based on the data, are shown as relative values.
10
Western Blot Analysis of EDN3 Protein
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Collect the cells from the cell flask into a 1.5 mL EP tube and added RIPA lysis buffer (Beyotime, CHN). The protein lysate (30 μg) was subjected to 10% SDS-PAGE and then electrotransferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore IPVH00010, Solarbio, CHN). The main antibodies are as follows: β-actin (ab6276, Abcam, UK), EDN3 (H00001908-M01, Novus Biologicals, USA), the ratios of them to the primary antibody dilution buffer (Beyotime, CHN) are 1:10000 and 1:1000. The bands were washed the next day and the secondary antibodies were incubated for one hour at room temperature. The ratio of β-actin and EDN3’s secondary antibody (Proteintech, CHN) to the secondary antibody dilution buffer (Beyotime, CHN) is 1:10000 and 1:5000. The bands were washed and soaked in ECL kit (yeasen, CHN) and analyzed by ChemiDoc XRS + image analyzer (Bio-Rad, USA).
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