Protein was extracted using SDS lysis buffer and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 1 h, they were incubated with primary antibodies overnight at 4 °C, which was followed by incubation with secondary antibodies for 2 h. Bands were visualized using a C-Digit Blot Scanner (Gene Company) and analyzed with ImageJ software. GAPDH (sc-47,724, 1:1000; Santa Cruz) was used as a protein loading control. Primary antibody against eIF4E (sc-9976, 1:500) was purchased from Santa Cruz.
C digit blot scanner
Manufactured by Gene Tech
Sourced in China
The C-Digit Blot Scanner is a laboratory equipment designed for the digitization and analysis of blot-based assays, such as Western blots, Northern blots, and Southern blots. The device captures high-resolution digital images of blot membranes and provides the necessary software tools for image processing and quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Eif4e, by Santa Cruz Biotechnology (2 mentions)
Supersignal west femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (2 mentions)
Sc 81178, by Santa Cruz Biotechnology (2 mentions)
Sc 8396, by Santa Cruz Biotechnology (2 mentions)
Ab22656, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Radioimmunoprecipitation assay buffer, by MedChemExpress (2 mentions)
Ab205719, by Abcam (2 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
0.45 m polyvinylidene fluoride membrane, by Merck Group (1 mentions)
β actin sc 1616 r, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Abcam (1 mentions)
Ripk1, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Sc 9976, by Santa Cruz Biotechnology (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
Ve cadherin, by Abcam (1 mentions)
5 protocols using c digit blot scanner
1
Protein Extraction and Western Blot
2
Cyclin D1 and β-Catenin Expression Analysis
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Protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) containing
50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.1% sodium dodecyl sulfate (SDS) was
added to the radioimmunoprecipitation assay buffer (pH = 8.0, MedChemExpress).
The protein was extracted by the addition of mixed solution to the cells. The
bicinchoninic acid protein quantification kit (Sigma-Aldrich Chemical Company)
was applied for protein quantification. Proteins were then subjected to 10%
SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 µm
polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA).
The membrane was sealed for 60 min with 5% bovine serum albumin and probed
overnight with primary antibodies to Cyclin D1 (sc-8396, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), β-catenin (ab22656, Abcam, Cambridge,
UK) and β-actin (sc-81178, Santa Cruz Biotechnology) at 4°C. The secondary goat
anti-mouse antibody (ab205719, Abcam) was used for a 1-h incubation at 25°C. The
immune response was detected using Super Signal West Femto Maximum Sensitivity
Substrate Kit (Thermo Fisher) and C-DiGit Blot Scanner (Gene Company, HK, China)
was used for image acquisition.
50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.1% sodium dodecyl sulfate (SDS) was
added to the radioimmunoprecipitation assay buffer (pH = 8.0, MedChemExpress).
The protein was extracted by the addition of mixed solution to the cells. The
bicinchoninic acid protein quantification kit (Sigma-Aldrich Chemical Company)
was applied for protein quantification. Proteins were then subjected to 10%
SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 µm
polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA).
The membrane was sealed for 60 min with 5% bovine serum albumin and probed
overnight with primary antibodies to Cyclin D1 (sc-8396, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), β-catenin (ab22656, Abcam, Cambridge,
UK) and β-actin (sc-81178, Santa Cruz Biotechnology) at 4°C. The secondary goat
anti-mouse antibody (ab205719, Abcam) was used for a 1-h incubation at 25°C. The
immune response was detected using Super Signal West Femto Maximum Sensitivity
Substrate Kit (Thermo Fisher) and C-DiGit Blot Scanner (Gene Company, HK, China)
was used for image acquisition.
3
Western Blot Protein Detection Protocol
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Protein was extracted using SDS lysis buffer and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 1 hour, they were incubated with primary antibodies overnight at 4°C, which was followed by incubation with secondary antibodies for 2 hours. Bands were visualized using a C‐Digit Blot Scanner (Gene Company) and analysed with ImageJ software. β‐actin (sc1616‐R, 1:1000; Santa Cruz) was used as a protein loading control. The antibodies are listed in Table S1 .
4
Western Blot Analysis of Cell Signaling
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Protein was extracted using SDS lysis buffer and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 1 h, they were incubated with primary antibodies overnight at 4 °C, which was followed by incubation with secondary antibodies for 2 hours. Bands were visualized using a C-Digit Blot Scanner (Gene Company) and analyzed with ImageJ software. GADPH (1:1000, sc-47724, Santa Cruz) was used as a protein loading control. The following primary antibodies were used: antibodies against eIF4E (1:200, sc-9976) from Santa Cruz, RIPK1 (1:500, ab106393), VE-cadherin (1:500, ab33168), vimentin (1:1000, ab92547), and Snail (1:1000, ab180714) from Abcam (Cambridge, USA), phospho-AKT (S473) (1:500, T40067F) from Abmart, AKT (1:500, #AF6259) from Affinity, E-cadherin (1:1000, #3195) from Cell Signaling Technology, and MMP-2 (1:500, 10373-2-AP) from Proteintech.
5
Western Blot Analysis of Cyclin D1 and β-Catenin
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Protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.1% sodium dodecyl sulfate (SDS) was added to the radioimmunoprecipitation assay buffer (pH = 8.0, MedChemExpress). The protein was extracted by the addition of mixed solution to the cells. The bicinchoninic acid protein quanti cation kit (Sigma-Aldrich Chemical Company) was applied for protein quanti cation. Proteins were then subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 µm polyvinylidene uoride membrane (Millipore Corporation, Billerica, MA, USA). The membrane was sealed for 60 min with 5% bovine serum albumin and probed overnight with primary antibodies to Cyclin D1 (sc-8396, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), β-catenin (ab22656, Abcam, Cambridge, UK) and β-actin (sc-81178, Santa Cruz Biotechnology) at 4℃. The secondary goat anti-mouse antibody (ab205719, Abcam) was used for a 1-h incubation at 25°C. The immune response was detected using Super Signal West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher) and C-DiGit Blot Scanner (Gene Company, HK, China) was used for image acquisition.
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