Immunoblotting was performed following standard procedures. Briefly, 10 μg of protein were separated on NuPAGE Novex Bis-Tris 4-12 % pre-cast gels (Invitrogen-Life Technologies (Carlsbad, CA, USA)) and transferred to Immobilon polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt; Germany). Unspecific binding was minimized by blocking the membranes for 1 h in 0.05 % Tween 20 (v/v in TBS) supplemented with 5 % w/v bovine serum albumin (Euromedex, Souffelweyersheim, France). Thereafter, membranes were probed with antibodies specific for TOMM20 (Abcam). Primary antibodies were revealed with suitable immunoglobulin G conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA), followed by chemiluminescence detection with the SuperSignal West Pico reagent in a ImageQuant 4000 (GE Healthcare, Little Chalfont, UK).
Nupage novex bis tris 4 12 pre cast gels
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels are polyacrylamide gels used for protein separation and analysis. These pre-cast gels provide a consistent and reproducible platform for electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Euromedex (3 mentions)
Immobilon polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Species specific immunoglobulin g conjugated to horseradish peroxidase, by Southern Biotech (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Mab1501, by Santa Cruz Biotechnology (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Horseradish peroxidase labeled secondary antibody, by GE Healthcare (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Dctm protein assay kit, by Bio-Rad (1 mentions)
Clone 8h10d10, by Cell Signaling Technology (1 mentions)
Na934, by GE Healthcare (1 mentions)
Clone 13e5, by Cell Signaling Technology (1 mentions)
Pierce ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab56053, by Abcam (1 mentions)
Ab2179, by Abcam (1 mentions)
Rabbit anti β actin antibody, by Merck Group (1 mentions)
10 protocols using nupage novex bis tris 4 12 pre cast gels
1
Immunoblotting of TOMM20 Protein
2
Immunoblotting analysis of reticulon-1c
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblotting, approximately 1 × 106 iRTN-1c MCA cells that had been incubated (or not, as control) with 0.3 µM tetracycline for 12–24 h, were washed with cold PBS, and lysed following standard procedures. Forty µg of proteins were separated according to molecular weight on NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen) and then electrotransferred to nitrocellulose membranes (Bio-Rad). Unspecific binding sites were blocked by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) and supplemented with 5% bovine serum albumin, followed by overnight incubation at 4 °C with primary antibodies specific for β-actin (MAB1501) or reticulon-1c (sc-71982, Santa Cruz Biotechnology). Primary antibodies were detected with the appropriate horseradish peroxidase-labeled secondary antibodies (Southern Biotechnologies Associates) and were revealed with the ECL Plus Western Blotting Detection System (GE Healthcare). The abundance of β-actin was monitored to ensure equal lane loading.
3
Immunoblotting Protocol for CFTR Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed following standard procedures. Briefly, 10 μg of protein were separated on NuPAGE Novex Bis-Tris 4–12% pre-cast gels (Invitrogen-Life Technologies, Carlsbad, CA, USA) and transferred to Immobilon polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany). Unspecific binding was reduced by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% w/v bovine serum albumin (Euromedex, Souffelweyersheim, France). Following, membranes were probed with antibodies specific for CFTR (Thermofisher) and beta-Actin (Abcam). Primary antibodies were revealed with species-specific immunoglobulin G conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA), followed by chemiluminescence analysis with the SuperSignal West Pico reagent by means of an ImageQuant 4000 (GE Healthcare, Little Chalfont, UK).
4
Immunoblotting for eIF2α and p-eIF2α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblotting, cells were washed with cold PBS at 4°C and lysed following standard procedures. Twenty μg of proteins were separated according to molecular weight on NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen, Waltham, USA) and electrotransferred to 9 Immobilon polyvinyldifluoride (PVDF) membranes (Millipore, Bedford, USA). Non-specific binding sites were blocked by incubating membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or BSA. After overnight incubation at 4°C, primary antibodies (rabbit polyclonal antibodies against eIF2α, and phospho-eIF2α (Ser51)) were detected with the appropriate horseradish peroxidase-labeled secondary antibodies (Southern Biotechnologies Associates; Birmingham; UK) and revealed with the Amersham ECL+ chemoluminescent substrate (GE Healthcare, Little Chalfont, UK). The abundance of β-actin was monitored to ensure equal lane loading.
5
Immunoblotting Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed following standard procedures. Cells were harvested and the obtained pellet was resuspended in RIPA buffer (89900; Thermo Fisher Scientific) supplemented with phosphatase and protease inhibitors (88669; Thermo Fisher Scientific) followed by sonication and protein content quantification by DCTM Protein Assay kit (5000112; Bio-Rad, Hercules, CA, USA). Then, 10 μg of protein were separated on NuPAGE Novex Bis-Tris 4–12% pre-cast gels (Invitrogen-Life Technologies, Carlsbad, CA, USA) and transferred to Immobilon polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany). Unspecific binding was reduced by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% w/v bovine serum albumin (Euromedex, Souffelweyersheim, France). Following, proteins were probed with antibodies specific for actin, LC3, mTOR, Phospho-mTOR (Ser2448), LAMP1, p70 S6 Kinase or SQSTM1/p62. Primary antibodies were revealed with species-specific immunoglobulin G conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA), followed by chemiluminescence analysis with the SuperSignal West Pico reagent by means of an ImageQuant 4000 (GE Healthcare, Little Chalfont, UK).
6
Quantifying Autophagy Markers in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of autophagy biomarkers in vivo, tissues were snap frozen, and then mechanically disrupted before cell lysis. For ATG7 detection, cell lines were subjected to standard lysis procedures. In both cases, 50 µg proteins were separated on NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen) and electrotransferred to polyvinyldifluoride (PVDF) membranes (Millipore Sigma). Membranes were blocked with 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk for 1 h and incubated overnight with primary antibody specific for MAP1LC3B (1:1000, #2775 from Cell Signaling Technology), SQSTM1 (1:1000, #5114 from Cell Signaling Technology), ATG7 (1:1000, clone D12B11, #8558 from Cell Signaling Technology; or 1:3000, clone ATG7-13, #SAB4200304 from Millipore Sigma), or ACTB (1:1000, clone 13E5, #4970 from Cell Signaling Technology; or 1:2000, clone 8H10D10, #3700 from Cell Signaling Technology), at 4 °C. Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-mouse (#NA931, from GE Healthcare Life Sciences, 1:5000) or anti-rabbit (#NA934, from GE Healthcare Life Sciences, 1:5000) secondary antibodies and revealed with the Pierce™ ECL Plus chemiluminescent substrate (#32132, Thermo Fisher) on a C600 Gel Doc & Western Imaging System operated by cSeries Capture v. 1.6.8.1110 (Azure Biosystems).
7
Immunoblot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in 8 M urea, 50 mM Tris HCl, pH 7.5 and 150 mM β-mercaptoethanol, sonicated and heated at 75°C for 10 minutes. Samples (equivalent of 2 x 105 cells) were subjected to electrophoresis in NuPAGE Novex 4–12% Bis-Tris pre-cast gels (Life Technologies). The procedures used for gel electrophoresis and immunoblotting have been described elsewhere [16 (link)]. Primary and secondary antibodies were used at the following concentrations: rabbit anti-BLM antibody (1:5,000; ab2179 from Abcam); rabbit anti-CDA antibody (1:500; ab56053 from Abcam); rabbit anti-β-actin antibody (1:10,000; Sigma); rabbit anti-PARP-1 antibody (1:4,000; ALX-210-302 from Enzo Life Sciences); rabbit anti-Chk2 (1/500; 2662 from Cell Signaling; rabbit anti-Chk2 T68 (1/500; 2661 from Cell Signaling; rabbit anti-H2AX (1/500; 2595 from Cell Signaling); rabbit anti-H2AX S139 (1/500; 2577 from Cell Signaling); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology).
8
Recombinant Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 3
TriA and variants thereof were produced in E. coli BL21(DE3) Gold (Agilent Technologies, Germany). Therefore E. coli was transformed with appropriate pET24d N-HIS tag expression vector and chaperone plasmid pGro7 (chaperones groEL and groES). Bacterial strains were grown at 30° C. in 100 mL LB for 20 h and protein expression induced with 0.1 mM IPTG at 25° C. for 20 hs. Cells were harvested by centrifugation at 3000 rpm at 4° C. for 20 min, resuspended in Bug Buster protein extraction reagent (Novagen, Germany) according to manufactures instructions. Lysates were clarified by centrifugation. Samples of bovine serum albumin (5, 10, and 20 g) were loaded onto each gel analyzed by densitometry to provide an internal standard. Protein determinations were verified using Coomassie protein assay dye, according to manufactures instruction (Thermo Scientific; USA). The HIS-tagged enzymes were purified by metal ion affinity chromatography using Ni-IDA 1000 kit (Macherey-Nagel, Germany) following manufactures instructions. Protein purity was accessed by SDS-PAGE using NuPAGE Novex 4-12% Bis-Tris pre-cast gels (Life Technologies; USA) stained with Coomassie Brilliant Blue (Serva, Germany). Protein concentrations were estimated by measuring absorbance at 280 nm using Lambda Bio+ (Perkin Elmer, USA).
9
Quantitative Western Blot Analysis
10
Optimizing Whole-Cell Lysis and Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared using RIPA buffer containing 1 mM dithiothreitol, 1 mM sodium orthovanadate, 10 μL/mL of EDTA-free, protease inhibitor cocktail (Sigma), 1 mM PMSF and 100 nM okadaic acid. After addition of lysis buffer cocktail, samples were rotated at 4°C for 30 minutes and subsequently passed through a syringe with a 27G needle five times. Samples were then centrifuged for five minutes at 13,000 rpm in a microcentrifuge to remove debris and supernatant collected. Protein was quantitated using a Bradford assay (BioRad). Absorbance was measured using a BioRad SmartSpec 3000.
For Western blot analysis, 60 μg of cell extracts were loaded into Nu-PAGE Novex 4–12% Bis-Tris pre-cast gels (Life Technologies). Protein was immobilized onto PVDF membrane, 0.45 μm pore size (Life Technologies). Blots were blocked for one hour with 3% (weight/volume) bovine serum albumin (Fisher Scientific) and incubated overnight at 4°C with a rabbit monoclonal antibody to RHAMM [EPR4055] antibody at 1:1,000 dilution (Abcam, Cambridge, MA. Catalogue number: ab108339). The next day blots were washed 4 times for 10 minutes with TBST and incubated for one hour at room temperature with an anti-rabbit secondary antibody at 1:5,000 dilution (R&D Systems). Bands were detected with enhanced chemiluminescence (Fisher Scientific).
For Western blot analysis, 60 μg of cell extracts were loaded into Nu-PAGE Novex 4–12% Bis-Tris pre-cast gels (Life Technologies). Protein was immobilized onto PVDF membrane, 0.45 μm pore size (Life Technologies). Blots were blocked for one hour with 3% (weight/volume) bovine serum albumin (Fisher Scientific) and incubated overnight at 4°C with a rabbit monoclonal antibody to RHAMM [EPR4055] antibody at 1:1,000 dilution (Abcam, Cambridge, MA. Catalogue number: ab108339). The next day blots were washed 4 times for 10 minutes with TBST and incubated for one hour at room temperature with an anti-rabbit secondary antibody at 1:5,000 dilution (R&D Systems). Bands were detected with enhanced chemiluminescence (Fisher Scientific).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!