Total PBMCs were stimulated with anti-CD3 (OKT3, 1ug/mL; eBioscience; 16–0037-85) for 48 hours. Cells were washed with PBS once and lysed in lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 2mM EDTA, 0.5% Triton X-100 and 0.5% NP40 and halt protease and phosphatase inhibitor cocktail [Sigma]) and samples were adjusted to have equal concentration of total protein and subjected to immunoblotting. Cell lysates were prepared and analyzed with anti-phospho-NFKB2 (S866/S870, Cell signaling #4810) and anti-NFKB2 (Cell Signaling Technology #3017) as previously described [6 (link)]. For the T cells blasts, total PBMCs were stimulated with anti-CD3 and anti-CD28 (1ug/mL; eBioscience) in the presence of IL-2 (10 ng/ml, Cell Signaling Technology) and cultured for 6–7 days. IL-2 was added every 2–3 days.
Halt protease and phosphatase inhibitor cocktail
Manufactured by Merck Group
Halt Protease and Phosphatase Inhibitor Cocktail is a lab equipment product designed to inhibit the activity of proteases and phosphatases in biological samples. It is formulated to help preserve the integrity of proteins and phosphorylated molecules during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin beads, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Anti cd3 and anti cd28, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Cell Signaling Technology (1 mentions)
Anti nfkb2, by Cell Signaling Technology (1 mentions)
Anti cd3 okt3, by Thermo Fisher Scientific (1 mentions)
Nuclear extraction kit, by Thermo Fisher Scientific (1 mentions)
Restriction enzymes, by Roche (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Syn per reagent, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rabbit anti h3, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Merck Group (1 mentions)
Anti flag horseradish peroxidase hrp, by Merck Group (1 mentions)
8 protocols using halt protease and phosphatase inhibitor cocktail
1
Investigating NF-κB2 Activation in T Cells
2
NB Cell Line Culture Protocol
3
Hippocampal Tissue Fractionation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For whole-cell lysate preparation, flash-frozen dorsal hippocampal tissue was homogenized mechanically in 300 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with 0.2% Triton X-100 (Sigma-Aldrich) and Protease and Phosphatase Inhibitor Cocktail (1:100; Thermo Fisher Scientific). The lysate was kept on ice for 30 min, following which they were centrifuged at 10,000g for 15 min at 4°C. The pellet was discarded, and the supernatant (whole-cell lysate) was collected for Western blot analysis. For synaptosomal extraction, hippocampal tissue was mechanically homogenized in Syn-PER Reagent (Thermo Fisher Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (1:100; Thermo Fisher Scientific). The homogenate was centrifuged at 1200g for 10 min at 4°C, after which the pellet (nuclear fraction) was discarded and the supernatant was centrifuged again at 15,000g for 25 min at 4°C. This pellet (synaptosomal fraction) was resuspended in RIPA buffer (Sigma-Aldrich) containing Halt Protease and Phosphatase Inhibitor Cocktail (1:100) and 0.2% Triton X-100 (Sigma-Aldrich).
4
Analyzing Nuclear Protein Dynamics via Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded and incubated overnight in culture media. Cells were lysed in Laemmli buffer supplemented with beta-mercaptoethanol and HALT protease and phosphatase inhibitor cocktail (Sigma). Isolation of nuclei from whole-cell lysis adapted a standard centrifugation-based protocol (79 (link)). Briefly, cells were lysed in 0.1% Igepal in PBS supplemented with phosphatase inhibitor cocktail. Nuclear fractions were sedimented by centrifugation. Whole-cell lysates and nuclear fraction were resolved using SDS-PAGE. Western blot was done using a standard protocol, and images were processed and quantified using ImageJ (https://imagej.net/ij/download.html ). For phospho-NF-kB experiments cells were serum starved for 4 to 8 h before lysing. Recombinant human TNF-α (R&D systems 210-TA) treatment was done at 20 ng/ml for 15 min before lysing cells. Recombinant human IFN-β (R&D systems 8499-IF-010) was done at 1000 IU/ml for 24 h before lysing cells. Antibodies used include: rabbit anti-JADE3 (Abcam #129495) used at 1:750 to 1:1000, rabbit anti-IFITM3 (Cell Signaling #59212) used at 1:750 to 1:1000, rabbit anti-NF-kB p65 (Cell Signaling #8242) used at 1:1000, rabbit anti-phospho-NF-kB p65 Ser536 (Cell Signaling #3033) used at 1:1000, rabbit anti-H3 (Cell Signaling), anti-Flag horseradish peroxidase (HRP) (Sigma), anti-rabbit IGG HRP (Sigma), and anti-GAPDH HRP (Sigma).
5
Subcellular Fractionation and Western Blotting of HuH7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates from human HuH7 hepatocytes were prepared in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA and 50 mM sodium fluoride) in the presence of protease and phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Sigma). For subcellular-fractionation and separation of cytoplasmic and nuclear proteins, cells were briefly washed with cold PBS and gently scraped with cytoplasmic protein extraction buffer (20 mM HEPES, 10 mM potassium chloride, 5 mM magnesium chloride, 1 mM EDTA, 10% glycerol and 0.2% NP-40). The cell lysates were centrifuged for 10 min at 775g and the supernatant collected for cytoplasmic proteins. Nuclear pellets were washed once with the cytoplasmic protein extraction buffer and lysed in RIPA buffer. Equal amounts of protein (10–100 μg) were resolved by 10% SDS-PAGE for western analysis. The primary antibodies for human phospho-Jnk (Thr183/Tyr185) (#4671S), total Jnk (#9252S), phospho AMPKα (Thr172) (#2531), total AMPKα (#2532S), and β-tubulin (#2146S) were purchased from Cell Signaling. The SREBP1/SREBP-1c antibody used was purchased either from Santa Cruz (sc-365513) for mouse liver lysates or Novus Biologicals (NB100-60545) for HuH7 cell lysates. The Lamin A antibody was purchased from Santa Cruz (sc-20680).
6
Western Blot and Affinity Precipitation of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HBEC3-KT cells or MLE-15 cells were suspended in NP-40 lysis buffer containing Halt protease and phosphatase inhibitor cocktail (Millipore), disrupted by sonication, and extracted at 4°C for 30 min. The protein concentration of the lysate was determined using bicinchoninic acid (BCA) protein assay (Pierce). 50 μg protein in 1× Laemmli buffer was separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were blotted with primary antibodies, detected by secondary antibodies with conjugated horseradish peroxidase, and developed using a Pico-sensitive chemiluminescence kit (Pierce). All membranes were stripped and re-blotted for β-actin or GAPDH as loading control.
Whole cell or mitochondria lysates were prepared with biotinylated ODN-treated HBEC3-KT cells or MLE-15 cells. To precipitate proteins Bound by biotinylated ODN in vivo, streptavidin beads (Pierce) were incubated with whole cell or mitochondria lysates containing 300 μg protein overnight at 4° C under constant gentle rotating. After incubation, streptavidin beads were centrifuged, washed with 1× PBS containing 0.05% Tween-20, resuspended in 50 μl of 2× SDS loading buffer, and boiled for 10 minutes. Elutes from the streptavidin beads were loaded onto SDS PAGE gel (Bio-Rad) and immunoblotted with VDAC1 or ANT1 antibody.
Whole cell or mitochondria lysates were prepared with biotinylated ODN-treated HBEC3-KT cells or MLE-15 cells. To precipitate proteins Bound by biotinylated ODN in vivo, streptavidin beads (Pierce) were incubated with whole cell or mitochondria lysates containing 300 μg protein overnight at 4° C under constant gentle rotating. After incubation, streptavidin beads were centrifuged, washed with 1× PBS containing 0.05% Tween-20, resuspended in 50 μl of 2× SDS loading buffer, and boiled for 10 minutes. Elutes from the streptavidin beads were loaded onto SDS PAGE gel (Bio-Rad) and immunoblotted with VDAC1 or ANT1 antibody.
7
Immunoblot Analysis of Influenza A M2 and DUOX
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblot assays, samples were exposed to lysis buffer in the presence of Halt protease and phosphatase inhibitor cocktail (Millipore), and the protein concentration in lysate was determined in a bicinchoninic acid protein assay. Thirty-five micrograms of protein in 2× Laemmli buffer was separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. The blots were probed with influenza A virus M2 (14C2), DUOX1 (H-9; Santa Cruz Biotechnology), and DUOX2 (AP11227c; Abgent, San Diego, CA) primary antibodies, detected by secondary antibodies with conjugated horseradish peroxidase, and developed using a Pico-sensitive chemiluminescence kit (Pierce). All membranes were stripped and probed for β-actin as the loading control. Densitometric semiquantitation of the protein band intensities was performed using ImageJ.
8
Western Blot and Affinity Precipitation of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HBEC3-KT cells or MLE-15 cells were suspended in NP-40 lysis buffer containing Halt protease and phosphatase inhibitor cocktail (Millipore), disrupted by sonication, and extracted at 4°C for 30 min. The protein concentration of the lysate was determined using bicinchoninic acid (BCA) protein assay (Pierce). 50 μg protein in 1× Laemmli buffer was separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were blotted with primary antibodies, detected by secondary antibodies with conjugated horseradish peroxidase, and developed using a Pico-sensitive chemiluminescence kit (Pierce). All membranes were stripped and re-blotted for β-actin or GAPDH as loading control.
Whole cell or mitochondria lysates were prepared with biotinylated ODN-treated HBEC3-KT cells or MLE-15 cells. To precipitate proteins Bound by biotinylated ODN in vivo, streptavidin beads (Pierce) were incubated with whole cell or mitochondria lysates containing 300 μg protein overnight at 4° C under constant gentle rotating. After incubation, streptavidin beads were centrifuged, washed with 1× PBS containing 0.05% Tween-20, resuspended in 50 μl of 2× SDS loading buffer, and boiled for 10 minutes. Elutes from the streptavidin beads were loaded onto SDS PAGE gel (Bio-Rad) and immunoblotted with VDAC1 or ANT1 antibody.
Whole cell or mitochondria lysates were prepared with biotinylated ODN-treated HBEC3-KT cells or MLE-15 cells. To precipitate proteins Bound by biotinylated ODN in vivo, streptavidin beads (Pierce) were incubated with whole cell or mitochondria lysates containing 300 μg protein overnight at 4° C under constant gentle rotating. After incubation, streptavidin beads were centrifuged, washed with 1× PBS containing 0.05% Tween-20, resuspended in 50 μl of 2× SDS loading buffer, and boiled for 10 minutes. Elutes from the streptavidin beads were loaded onto SDS PAGE gel (Bio-Rad) and immunoblotted with VDAC1 or ANT1 antibody.
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!