T cells, unstimulated or stimulated for 24 hours (mouse) or 48 hours (human) with anti-CD3 (4μg/ml) and CD28 (2μg/ml) with or without chloroquine (10μM) or 3-MA (5 mM) were harvested and washed twice with PBS. Then whole cells were lysed in RIPA lysis extraction buffer (Thermo Scientific). Equal amounts of protein lysates quantified with the BCA Protein Assay Kit (Thermo Scientific) were separated with 10% or 12.5% SDS-PAGE. Rabbit-LC3 antibody (Novus Biologicals, Littelton, CO) was used according to manufacturer’s instructions. Secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) was used to detect the primary antibody. Densitometric analysis was performed using ImageJ software.
Ripa lysis extraction buffer
Manufactured by Thermo Fisher Scientific
RIPA lysis extraction buffer is a solution used to extract and solubilize proteins from cells and tissues. It contains a combination of detergents, salts, and other components that help to disrupt cell membranes and release the proteins within. The buffer is commonly used in Western blotting, immunoprecipitation, and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Odyssey clx imaging system, by LI COR (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protease phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Image studio lite 5, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Rabbit lc3 antibody, by Novus Biologicals (1 mentions)
Nb100 417, by Novus Biologicals (1 mentions)
Pa1 24894, by Thermo Fisher Scientific (1 mentions)
Nb110 39115, by Novus Biologicals (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Tween 20, by Beyotime (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
8 protocols using ripa lysis extraction buffer
1
Immunoblotting of autophagy markers
2
Myosin and Actin Fractionation from Muscle Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The myosin and actin fractionation methods were conducted, with slight modifications, as described by Roberts et al. (54 (link)). In brief, 20 mg of dorsal muscle tissue was dissected and immediately submerged in 500 µL of homogenization buffer (98% RIPA lysis extraction buffer (ThermoFisher #89900), 1% Halt protease/phosphatase inhibitor cocktail (Thermo Scientific #78442), 1% EDTA). The tissue was homogenized with triple pure M-Bio grade high-impact zirconium beads in a Beadbug 6 microtube homogenizer bead beater at 4,900 rpm for 60 s followed by gentle agitation at 4 °C for 60 min. The sample was centrifuged at 4 °C at 3,000 × g for 30 min. The supernatant (cytosolic fraction) was removed and frozen at −80 °C. The pellet was resuspended with 500 µL of homogenization buffer, centrifuged for 10 min at 3,000 × g, and the supernatant was removed followed by two additional washes. The remaining myofibrillar pellet was then immediately frozen and placed at −80 °C until further processing. Electrophoresis of the sarcoplasmic and myofibrillar fractions is detailed in SI Appendix, Supplementary Methods .
3
Protein Extraction and Immunoblotting from Adipose Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from adipose tissues were extracted by a RIPA lysis/extraction buffer (89900, Thermo Fisher Scientific) containing a Halt Protease Inhibitor Cocktail (87785, Thermo Fisher Scientific). An equal amount of protein from each sample was loaded onto Mini-PROTEAN TGX Gels (4561086, Bio-Rad) and transferred to PVDF membranes (1620184, Bio-Rad). Immunoblotting was incubated with the primary antibodies specific for UCP1 (1:1,000 dilution, PA1-24894, Thermo Fisher Scientific), COX4 (1:1,000 dilution, NB110-39115, Novus Biologicals), CA9 (1:1,000 dilution; NB100-417; Novus Biologicals) and HIF1α (1:1,000 dilution, 36169, Cell Signaling Technology). A primary antibody against β-actin (1:2,000 dilution, 3700, Cell Signaling Technology) was used to justify the sample loading levels. Secondary antibodies-conjugated with IRDye 680RD donkey anti-mouse (1:15,000 dilution, 926-68072, LI-COR Biosciences) and IRDye 800CW donkey anti-rabbit (1:15,000 dilution, 926-32213, LI-COR Biosciences) were incubated. Densitometry analysis was performed using the Odyssey CLX Imaging System (LI-COR Biosciences).
4
Muscle Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately, 30 mg of frozen skeletal muscle was submerged in an ice-cold RIPA lysis extraction buffer (ThermoFisher #89900) supplemented with 1% Halt protease/phosphatase inhibitor cocktail (Thermo Scientific #78442) and homogenized with triple pure M-Bio grade high impact zirconium beads in a Beadbug 6 microtube homogenizer. Muscle lysate was gently agitated at 4 °C for 2 h and spun down at 16,000 × g for 20 min. The subsequent supernatant was collected and snap frozen in liquid nitrogen. Protein quantification was determined with a Pierce BCA Protein Assay Kit (Thermo #23227). SDS-Page and western blotting for pAMPK (T172) (CST #40H9) and pAb TOM20 (11802-1-AP) can be found in SI Appendix, Supplementary Methods .
5
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein cleavage extraction solution was made using RIPA lysis extraction buffer (Thermo Fisher Scientific, 89901) and protease phosphatase inhibitors (Thermo Fisher Scientific, A32953). The hippocampus tissues were mixed with the protein cleavage extraction solution and homogenized. Nanodrop (Thermo Scientific) was used to measure the protein concentration of each group. Before running the gel, the proteins were boiled at 95°C for 5 min to denature. Proteins were separated by 8% SDS-PAGE and transferred onto PVDF membranes (0.45 μm; Millipore, Billerica, USA). Membranes were placed in 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 30 min at room temperature to block nonspecific binding sites before incubating overnight at 4°C with primary antibody solution. On the second day, TBST was used to wash the membranes for 8 mins/3 times, followed by incubation with corresponding IRDye 680RD secondary antibodies in TBST for 2h at room temperature. After following 8 mins/3 times wash with TBST, the immunoreactive bands were scanned and captured by the Odyssey CLx Imaging System (LI-COR Biosciences) and quantitatively analyzed by densitometry with Image Studio Lite 5.2 (LI-COR Biosciences).
6
NSCLC Cell Protein Extraction and ABCB1 Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis extraction buffer (Thermo Fisher Scientific, Inc.) was used for total protein extraction from NSCLC cells and protein concentration was determined by performing a bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Subsequently, 8-12% SDS-PAGE was used to separate protein samples (25 µg), and then the samples were transferred on PVDF membranes (EMD Millipore). The membrane was blocked in PBS containing 0.1% Tween-20 (Beyotime Institute of Biotechnology) and 5% non-fat dry milk at room temperature for 2 h. The membranes were first incubated with the following primary antibodies: Anti-ABCB1 (1:1,000; cat. no. 13342; Cell Signaling Technology, Inc.) or anti-GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) for overnight incubation at 4˚C. The membranes were then incubated with horseradish peroxidase-anti-rabbit secondary antibody (1:2,000; cat. no. 14708; Cell Signaling Technology, Inc.) for 1 h at room temperature. Finally, the protein bands were detected using ECL solution (Pierce; Thermo Fisher Scientific, Inc.) and images were captured using a FluorChem imaging system (ProteinSimple) GADPH was used as a loading control.
7
Protein Expression Analysis in Mouse Lungs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mouse lungs or MLE-12 Cells were lysed by RIPA Lysis Extraction Buffer (catalog #89901; Thermo Fisher Scientific) along with protease and phosphatase inhibitors cocktail (catalog #78445; Thermo Fisher Scientific). Total protein concentration was determined by Pierce BCA protein assay reagent kit (catalog #23227; Thermo Fisher Scientific) according to the manufacturer’s protocol. 40 μg of total protein was loaded and separated by SDS-polyacrylamide gel electrophoresis. The gel was transferred using a Mini Trans-Blot cell (Biorad) to PVDF membrane (catalog #IPVH00010; EMD Millipore). Proteins were detected by mouse anti-IGFBP2 (R&D Systems, catalog #MAB7971), anti-P21 (Abcam, catalog #ab188224), anti-β-actin (catalog #sc-47778HRP; Santa Cruz Biotechnology) and anti-histone H3 (catalog #4499; Cell Signaling Technology). Immunoblots were incubated with SuperSignal™ West Pico or Femto Maximum Sensitivity Substrate (catalog #34095; Thermo Fisher Scientific).
8
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein cleavage extraction solution was made using RIPA lysis extraction buffer (Thermo Fisher Scientific, 89901) and protease phosphatase inhibitors (Thermo Fisher Scientific, A32953). The hippocampus tissues were mixed with the protein cleavage extraction solution and homogenized. Nanodrop (Thermo Scientific) was used to measure the protein concentration of each group. Before running the gel, the proteins were boiled at 95°C for 5 min to denature. Proteins were separated by 8% SDS-PAGE and transferred onto PVDF membranes (0.45 μm; Millipore, Billerica, USA). Membranes were placed in 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 30 min at room temperature to block nonspecific binding sites before incubating overnight at 4°C with primary antibody solution. On the second day, TBST was used to wash the membranes for 8 mins/3 times, followed by incubation with corresponding IRDye 680RD secondary antibodies in TBST for 2h at room temperature. After following 8 mins/3 times wash with TBST, the immunoreactive bands were scanned and captured by the Odyssey CLx Imaging System (LI-COR Biosciences) and quantitatively analyzed by densitometry with Image Studio Lite 5.2 (LI-COR Biosciences).
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!