Whole cell lysates were obtained by resuspension of cell pellets in 500 μl RIPA buffer (Sigma, USA) and 40 μl of protease inhibitor cocktail (Sigma, USA). The lysates were estimated for protein concentration using BCA Assay method. Protein extracts (40 μg) were subjected to SDS-PAGE using 12% Tris/HCl SDS gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Membrane Technologies, India). Membranes were blocked using 3% BSA (Himedia, India) for 2 hours, followed by incubation with primary antibody against β- actin (Abcam,UK, 1:500) and tyrosine hydroxylase (Santa Cruz, USA, 1:500) in 1% BSA- phosphate saline buffer (PBS) overnight at 4°C. Post incubation, membranes were washed thrice in TBST and incubated with the appropriate horseradish peroxidase (HRP) - conjugated secondary antibody (1/4,000) (Dako, USA) for 2 hours at room temperature. Membranes were developed with chemiluminescence detection reagents (Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).
Gel imager machine fluor chem e
Manufactured by Cell Biosciences
Sourced in Australia
The Gel Imager machine (Fluor Chem E) is a laboratory equipment used for capturing and analyzing gel electrophoresis images. It provides high-resolution imaging capabilities for various types of gel-based assays, such as protein and nucleic acid gels.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
β actin, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by HiMedia (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Primary antibody against β actin, by Abcam (1 mentions)
Tyrosine hydroxylase, by Santa Cruz Biotechnology (1 mentions)
Ab8227, by Santa Cruz Biotechnology (1 mentions)
4 protocols using gel imager machine fluor chem e
1
Quantitative Western Blot Analysis
2
Immunoblotting for Neuronal Cell Proteins
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Immunoblotting for the expression of neuronal cell specific proteins was performed with both induced and uninduced control cells, as previously described18 (link). Briefly, after preparing whole cell lysates using RIPA buffer (Sigma, USA), the protein quantification was done using Bicinchoninic Acid (BCA) Assay method. Protein extracts (30 μg) were subjected to SDS-PAGE using 12% Tris/HCl SDS (Sodium dodecyl sulphate) gels and transferred onto PVDF membranes (Membrane Technologies, India). After blocking the membranes with 3% BSA, they were incubated with primary antibody against β-actin (Abcam,UK, 1:2500), MAP-2 (Santa Cruz, USA, 1:1000) and tyrosine hydroxylase (Santa Cruz, USA, 1:1000) in 1% BSA- phosphate saline buffer (PBS) overnight at 4 °C. Post incubation, membranes were washed thrice in PBST and incubated with the appropriate horseradish peroxidase (HRP)- conjugated secondary antibody (1/4,000) (Dako, USA) for 2 hours at room temperature. Membranes were developed with chemiluminescence detection reagent (Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).
3
Immunoblotting Analysis of Neuronal Proteins
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Immunoblotting for the expression of neuronal cell-specific proteins was performed as previously described [13 (link)]. Briefly, after preparing whole cell lysates using RIPA buffer (Sigma), BCA Assay method was used for protein quantification. Protein extracts (30 μg) were subjected to SDS-PAGE using 12% Tris/HCl SDS (Sodium dodecyl sulphate) gels and transferred onto PVDF membranes (Membrane Technologies, Ambala, India). 3% BSA at room temperature was used to block the membranes. They were then incubated with primary antibodies against β-actin (Abcam, 1:2500), MAP-2 (Abcam, 1:1500) and TH (Santa Cruz Biotech., Heidelberg, Germany, 1:1000) in 1% BSA-phosphate saline buffer (PBS) at 4 °C overnight. After incubation followed by washing thrice with TBS, membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1/4000) (Dako, Santa Clara, CA, USA) at room temperature for 2h. Membranes were developed with chemiluminescence detection reagent (Pierce, WA, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Preston, VIC, Australia).
4
Immunoblotting for Neuronal Protein Expression
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+ Expand
Immunoblotting for the expression of neuronal cell-specific proteins was performed with both induced and uninduced control cells, as previously described7 (link). Briefly, after preparing whole cell lysates using RIPA buffer (#R0278, Sigma, USA), the protein quantification was done using bicinchoninic acid (BCA) assay method, according to the manufacturer’s protocol (#00-4333-57, Pierce, ThermoScientific, USA). Protein extracts (30 μg) were subjected to SDS–PAGE using 12% Tris/HCl sodium dodecyl sulphate (SDS) gels and transferred onto PVDF membranes (Membrane Technologies, India). After blocking the membranes with 3% BSA, they were incubated with primary antibody against β-actin (1:2500, #ab8227), MAP-2 (1:1500) and TH (1:1000, #sc-73151, Santa Cruz, USA) in 1% BSA-phosphate saline buffer (PBS) overnight at 4 °C. Post incubation, membranes were washed thrice in PBST and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1/4000) (Dako, USA) for 2 h at RT. Membranes were developed with chemiluminescence detection reagent (#34580, Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).
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