Cell extracts were prepared by lysing cells with RIPA buffer (Thermo Scientific). Samples were sonicated and cleared by centrifugation (1,200 rpm) at 4℃ for 10 minutes. Supernatant protein concentrations were determined by BCA protein assay reagent (Thermo Scientific). Samples containing equal amounts of protein were separated by NuPAGE 4% to 12% Bis-Tris gels (Invitrogen), followed by transfer of resolved proteins to nitrocellulose membranes using the Xcell SureLock electrophoresis system (Invitrogen). Nonspecific binding was blocked for 1 hour at room temperature. Blots were then probed overnight at 4℃ with primary antibodies, followed by 1-hour incubations with secondary antibodies conjugated to goat anti-rabbit horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA), and then developed by chemiluminescence detection using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Quantification of the signals was performed using a Gel Doc XR+ system (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard.
Xcell surelock electrophoresis system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Xcell SureLock electrophoresis system is a laboratory equipment designed for the separation and analysis of macromolecules, such as proteins and nucleic acids, using gel electrophoresis. The system provides a controlled and consistent environment for the electrophoretic separation process.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Gel doc xr system, by Bio-Rad (3 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (3 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Protran nitrocellulose membrane, by Cytiva (2 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions)
Whatman, by Cytiva (2 mentions)
Protran, by Cytiva (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Simplyblue, by Thermo Fisher Scientific (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
E cadherin, by BioLegend (1 mentions)
Clone 36 e cadherin, by BD (1 mentions)
11 protocols using xcell surelock electrophoresis system
1
Immunoblot Analysis of Protein Expression
2
Western Blot Protein Detection Protocol
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Reducing agent and loading buffer were added to protein extracts followed by heat treatment for 10 min at 95°C. Samples were centrifuged at 4°C for 10 min at 13,000 g and loaded onto a 10% Bis-Tris pre-cast gel. Protein were separated at 100 V for 45 min in 1x NuPAGE MES running buffer using the Invitrogen XCell SureLock electrophoresis system. 4 μl of PageRuler prestained marker was used to estimate protein size. Gels were transferred onto PVDF membrane (Amersham) and blocked with 5% BSA in TBST for 1 h at room temperature. Membranes were incubated with primary antibody in TBST containing 5 % BSA over night at 4°C. Antibody concentrations used were as follows: anti-Burs (Scopelliti et al., 2016 (link)) (1:500) and anti- αTub (1:1,000). Membranes were washed 3 times for 15 min in TBST and incubated with fluorescent IRDye (680RD and 800CW) secondary antibody (1:10,000) for 2 h at room temperature in 5% BSA/TBST. Membranes were washed and blots were visualised using the LiCor ODYSSEY Clx. Fluorescent intensity was measured using the Fiji software.
3
Western Blot Protein Quantification
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Cell extracts were prepared by lysing cells with RIPA buffer (Thermo Scientific, Carlsbad, CA, USA). Samples were sonicated and cleared by centrifugation (1,200 rpm) at 4℃ for 10 minutes. Supernatant protein concentrations were determined using BCA protein assays (Thermo Scientific, Carlsbad, CA, USA). Samples containing equal amounts of protein were separated by NuPAGE 4–12% Bis-Tris gels (Invitrogen), followed by transfer of resolved proteins to nitrocellulose membranes using the XCell SureLock electrophoresis system (Invitrogen). Nonspecific binding was blocked for 1 hour at room temperature. Blots were then probed overnight at 4℃ with primary antibodies, followed by 1-hour incubation with secondary antibodies conjugated to goat anti-rabbit horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Western blots were developed using Super Signal West Pico Chemiluminescent substrate (Thermo Scientific). Quantification of the signals was performed using the Gel Doc XR+ system (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard.
4
Protein Characterization by SDS-PAGE and Western Blotting
5
SDS-PAGE and Western Blot Analysis of Membrane Proteins
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Protein characterization was carried out using the SDS-PAGE method. The cracked CC membrane samples were suspended in lithium dodecyl sulfate loading buffer (Invitrogen). Samples were heated to 90°C for 10 min, and 20 μl of sample was loaded into each well of a NuPAGE Novex 4 to 12% bis-tris minigel, using Mops SDS as the running buffer (Invitrogen) in an XCell SureLock Electrophoresis System based on the manufacturer’s instructions. Protein staining was accomplished using Coomassie Blue (Invitrogen) and destained in water overnight before imaging. For Western blot analysis, the protein was transferred to Protran nitrocellulose membranes (Whatman) using an XCell II Blot Module (Invitrogen) in NuPAGE transfer buffer (Invitrogen) per the manufacturer’s instructions. Membranes were probed using antibodies against CD44 (clone 515; BD Biosciences), E-cadherin (clone 36; BD Biosciences), and CD49e (BD Biosciences), followed by horseradish peroxidase–conjugated anti-mouse immunoglobulin G (Cell Signaling Technology) as the secondary antibody.
6
Protein Quantification and Western Blotting
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Cell extracts were prepared by lysing cells with RIPA buffer (Thermo Scientific). Samples were sonicated and cleared by centrifugation. Supernatant protein concentrations were determined by BCA protein assay reagent (Thermo Scientific). Samples containing equal amounts of protein were separated by NuPAGE 4% to 12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA), followed by transfer of resolved proteins to nitrocellulose membranes using the Xcell SureLock electrophoresis system (Invitrogen). Nonspecific binding was blocked for 1 hour at room temperature. Blots were then probed overnight at 4°C with primary antibodies, followed by 1-hour incubations with secondary antibodies conjugated to goat anti-rabbit horse-radish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA), and then developed by chemiluminescence detection using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Quantification of the signals was performed using a Gel Doc XR+ system (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard.
7
Protein Pulldown and Validation
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Samples were prepared and validation pulldowns were performed as described in “Protein extraction” and “RNA pulldown”, respectively. Proteins bound to RNA-baits were eluted in 50 µL of sample buffer (Biorad, 1610737) and boiled for 5 min at 95
oC in a heat block (Eppendorf 5355). Boiled samples were then spun at 13,000 rpm in a microcentrifuge (Eppendorf 5430R). Pulldown eluates (100%) and input controls (5%) were loaded alongside a protein ladder (PageRuler Plus pre-stained protein ladder 10–250 kDA, Thermo Fisher Scientific 26619) in a 4-12% Bis-Tris gel and ran in NuPAGE MOPS SDS running buffer (Invitrogen) at 200V for 50 min in a XCell SureLock electrophoresis system (Thermo Fisher Scientific) connected to a Bio-Rad PowerPac Universal Power Supply (Bio-Rad 1645070).
oC in a heat block (Eppendorf 5355). Boiled samples were then spun at 13,000 rpm in a microcentrifuge (Eppendorf 5430R). Pulldown eluates (100%) and input controls (5%) were loaded alongside a protein ladder (PageRuler Plus pre-stained protein ladder 10–250 kDA, Thermo Fisher Scientific 26619) in a 4-12% Bis-Tris gel and ran in NuPAGE MOPS SDS running buffer (Invitrogen) at 200V for 50 min in a XCell SureLock electrophoresis system (Thermo Fisher Scientific) connected to a Bio-Rad PowerPac Universal Power Supply (Bio-Rad 1645070).
8
Protein Lysate Electrophoresis and Immunoblotting
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Crude mitochondrial or total protein lysates were resuspended in the NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) and loaded onto precast NuPAGE 4 to 12% bis-tris gels (Thermo Fisher Scientific) in an XCell SureLock electrophoresis system (Thermo Fisher Scientific). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 5% (w/v) milk in tris buffer saline with 1% (v/v) Tween 20 (TBST; Sigma-Aldrich). Immunoblotting was performed following standard techniques and developed using Clarity Western ECL substrate (Bio-Rad).
9
Protein Extraction and Western Blot Analysis
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Total cell proteins were extracted using RIPA buffer supplemented with a protease inhibitor and phosphatase inhibitor cocktail. Protein concentrations were measured using the XL-Bradford kit (Aproscience, Tokushima, Japan). Tris-glycine SDS sample buffer was added to protein samples and boiled at 100 °C for 5 min. Ten micrograms of protein were loaded onto a 4–20% Tris-Glycine gel (Thermo Fisher Scientific, Waltham, MA, USA) and electrophoresed at 125 V for 60 min using the Invitrogen XCell SureLock electrophoresis system with Tris-Glycine SDS Running Buffer (Thermo Fisher Scientific). Proteins were then transferred onto PVDF membranes using the iBlot2 Dry Blotting System (Thermo Fisher Scientific). Membranes were blocked with 5% non-fat dried skimmed milk and incubated with primary antibodies, including anti-STING mAb (#13647, Cell Signaling Technology), anti-cGAS (#79978, Cell Signaling Technology), and anti-β-actin mAb (sc-69879, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. The membranes were then incubated with HRP-linked anti-mouse IgG or anti-rabbit IgG antibodies (Cell Signaling Technology) at room temperature for 1 h. Immunoreactive proteins were visualized using ImageQuant LAS 4000 mini (Fuji Film, Tokyo, Japan) with ECL prime Western blot detection reagent (GE Healthcare, Chicago, IL, USA).
10
Western Blot Analysis of Protein Samples
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Protein samples were electrophoresed using Novex® NuPAGE® 4–12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Thermo Fisher Scientific, Waltham, MA, USA, Product # EI0002) and Precision Plus Protein™ WesternC™ Protein Standards (Bio-Rad, Hercules, CA, USA, Product # 1610376) at 120 volts for 1 h and 30 min. Proteins were then transferred onto a PVDF Membrane (Thermo Fisher Scientific, Waltham, MA, USA, Product #LC2002) using the XCell II™ Blot Module (Thermo Fisher Scientific, Waltham, MA, USA, Product #EI9051) at 280 mV for 1 h 30 min. The membrane was blocked for one hour with 5% Bovine Serum Albumin (BP1600-100) at 25 °C. The membrane was probed with the relevant primary antibody overnight at 4 °C followed by washes in PBST and HRP conjugate secondary antibody incubation (Amersham ECL Western Blotting Detection Kit RPN2108, 1:2000 dilution) for 1 h at 25 °C. Chemiluminescent detection was performed using Amersham ECL Western Blotting Detection Kit RPN2108 (Sigma-Aldrich, Inc, St. Louis, MO, USA). Densitometry analysis of western blot was performed using Azure densitometrics™ (Azure biosystems, Dublin, CA, USA).
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