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Np0321box

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The NP0321BOX is a laboratory equipment item produced by Thermo Fisher Scientific. It is designed to serve a core function within a laboratory environment. The description of this product is limited to its factual and unbiased specifications, without any interpretation or extrapolation on its intended use.

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Lab products found in correlation

Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions) Azure c300 imaging system, by Azure Biosystems (3 mentions) Laemmli buffer, by Bio-Rad (3 mentions) β actin, by Merck Group (3 mentions) Ab129002, by Abcam (2 mentions) Ab123545, by Abcam (2 mentions) Ab203694, by Abcam (2 mentions) Ab110411, by Santa Cruz Biotechnology (2 mentions) Np0007, by Thermo Fisher Scientific (2 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions) P5726, by Merck Group (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Np0323box, by Thermo Fisher Scientific (2 mentions) Np0002, by Thermo Fisher Scientific (2 mentions) Image studio software version 5, by LI COR (1 mentions) Bradford reagent, by Merck Group (1 mentions) Protease inhibitor, by Roche (1 mentions) Phosphatase inhibitor, by Roche (1 mentions) Amersham hyperfilm ecl, by Cytiva (1 mentions) Ecl western blotting detection reagent, by Cytiva (1 mentions)

28 protocols using np0321box

1

Protein Extraction and Analysis from 3D Spheroids

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A total of 180 spheroids were collected and dissociated as described before. Samples were washed and lysed with 1x RIPA buffer (Bioconcept, Allschwil, Switzerland, CellSignaling, 9806S) supplemented with phosphatase inhibitor (Roche, Basel, Switzerland, 04906837001) and protease inhibitor (Roche, 11836170001) for ≥10 min on ice. The extract was centrifuged for 10 min at 14,000 rpm at 4 °C. The protein concentration was determined using the Bradford reagent (Sigma-Aldrich, B6916-500ML) and by analyzing the absorbance at 595 nm on a Biotek Cytation 3.
A quantity of 50 µg of total protein content was loaded on SDS-page gels (Thermofisher, Invitrogen, NP0321BOX). Proteins were separated based on their molecular weight through gel electrophoresis. The gel content was transferred onto a nitrocellulose membrane (Amersham Protran 10600007) and protein bands were identified through immunofluorescence staining (Table S5). The analysis was done on the LI-COR scanner and the intensity was analyzed using the ImageStudio software version 5.2 (LI-COR Biosciences, NE, USA).
Rausch M., Blanc L., De Souza Silva O., Dormond O., Griffioen A.W, & Nowak-Sliwinska P. (2021). Characterization of Renal Cell Carcinoma Heterotypic 3D Co-Cultures with Immune Cell Subsets. Cancers, 13(11), 2551.
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2

Western Blot Protocol for Protein Analysis

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Samples were mixed with 4 x LDS sample buffer (ThermoFisher NP0007) and 0.1M DTT. Samples were loaded onto a 4–12% Bis-Tris gel (Invitrogen NP0321BOX) in 1x MOPs buffer and run at 160V for 1 hour. The gel was transferred to a nitrocellulose membrane using the wet transfer method in 1x transfer buffer (25mM Tris base, 192mM glycine, 20% methanol) at 400mA for 75 minutes at 4°C. The membrane was blocked in a blocking buffer (5% non-fat milk powder in 1x Tris buffer saline with 0.1% Tween 20) for at least 60 minutes. Primary antibodies were diluted in blocking buffer and incubated with membranes overnight at 4°C. Membranes were washed 3 × 20 minutes with 1 x TBST, incubated for 1 hour at 25°C with secondary HRP-conjugated antibodies against rabbit IgG (Cell Signaling Technology, 7074S), washed again 3× 20 minutes with 1x TBST, and developed using ECL Western Blotting Detection Reagents (Cytiva) and Amersham Hyperfilm ECL (Cytiva) using an automated M35 X-OMAT Processor developer (Kodak).
McShane E., Couvillion M., Ietswaart R., Prakash G., Smalec B.M., Soto I., Baxter-Koenigs A.R., Choquet K, & Churchman L.S. (2023). A kinetic dichotomy between mitochondrial and nuclear gene expression drives OXPHOS biogenesis. bioRxiv.
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3

Western and Dot Blot Assay for Aβ Aggregation

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A total of 3 μl of each sample was incubated for 0 h, 12 h, and 24 h with 3 μl Nu-PAGE™ LDS Sample Buffer (Invitrogen) and 6 μl ddH2O. The mixture was electrophoresed in a Nu-PAGE 4%~ 12% Bis-Tris premade gel (Invitrogen NP0321BOX) at 200 V for 30 min, and the protein was transferred to a 0.2 μm PVDF membrane. The 6E10 antibody was used for Western blotting to detect Aβ aggregation. Another 2 μl of the sample was incubated for 24–120 h or was not incubated and was spotted on an NC membrane (Sangon) for dot blotting analysis to detect Aβ oligomers.
Zhang J.X., Lai Y.H., Mi P.Y., Dai X.L., Zhang R., Zhang Z.J., Zhang S.J., Zhang X.W., Zhang X.Y., Yang B.Y., Cui D.M., Zhang C., Zhao C.Q, & Dou F. (2019). Rescue of cognitive deficits in APP/PS1 mice by accelerating the aggregation of β-amyloid peptide. Alzheimer's Research & Therapy, 11, 106.
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4

In Vitro Methylation Assay of SETDB1

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In vitro methylation assays were performed using normalized amount of flag immunoprecipitated (with the beads) SETDB1 (WT or CA or NLS) from total extract of stable ES cell lines, 1 μg recombinant H3.1 (NEB M2503S and 2 μl Adenosyl-L-methionine, S-[methyl-3H] (Perkin Elmer NET155V250C) in 50mM Tris pH8,20 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol and incubated at 30°C for 2 hours. Samples were separated on 4-12% NUPAGE gels (Invitrogen NP0321BOX) in MOPS buffer (Invitrogen NP0001), Coomassie stained (Invitrogen LC6060). Then gel was dried and autoradiography performed on Amersham Hyperfilm (ref28906843) at -80°C.
Rapone R., Del Maestro L., Bouyioukos C., Albini S., Cruz-Tapias P., Joliot V., Cosson B, & Ait-Si-Ali S. (2023). The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells. iScience, 26(8), 107386.
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5

Western Blot and Capillary Electrophoresis Profiling

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Cells were lysed in the Laemmli buffer (Bio-Rad) containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher, 78440). For standard Western blot, samples were run on a 4%–12% SDS PAGE gel (Invitrogen, NP0321BOX) and were transferred to a polyvinylidene difluoride membrane. The western blots were captured by using the Azure (C300) imaging system (Azure Biosystems). Protein capillary electrophoresis was detected by using the Wes instrument (ProteinSimple). The antibodies were used in the standard Western blot: acetyl-histone H3 (Lys27; D5E4; CST 8173, 1:500), histone H3 (CST 14269; 1:500), PARP (CST 9532; 1:500), cCP9 (CST 7237; 1:500), cCP3 (CST 9665; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2,000), Mcl-1 (CST 5453; 1:500), Bcl-xL (CST 2764; 1:500), SDHA (Abcam, ab123545; 1:500), OXPHOS (Abcam, ab110411;1:500), ClpP (B-12; Santa Cruz Biotechnology Inc, sc-271284; 1:500), ClpX (Abcam, ab203694, 1:500), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Thermo Fisher, 31460), and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Thermo Fisher, 31430). The antibodies were used in the protein capillary electrophoresis: HDAC1 (CST 34589; 1:25), HDAC2 (CST 57156; 1:25), and Vinculin (Abcam ab129002, 1:500).
Nguyen T.T., Shang E., Schiffgens S., Torrini C., Shu C., Akman H.O., Prabhu V.V., Allen J.E., Westhoff M.A., Karpel-Massler G, & Siegelin M.D. (2022). Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma. Clinical Cancer Research, 28(9), 1881-1895.
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6

Western Blot and Capillary Electrophoresis

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Cells were lysed in the laemmli buffer (Biorad) containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher, 78440). For standard western blot, samples were run on a 4-12% SDS PAGE gel (Invitrogen, NP0321BOX) and were transferred to a PVDF membrane. The western blots were captured by using the Azure (C300) imaging system (Azure Biosystems). Protein capillary electrophoresis was detected by using the Wes instrument (ProteinSimple, CA). The antibodies were used in the standard western blot: acetyl-histone H3 (Lys27) (D5E4) (CST 8173, 1:500), histone H3 (CST 14269; 1:500), PARP (CST 9532; 1:500), cCP9 (CST 7237; 1:500), cCP3 (CST 9665; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2,000), Mcl-1 (CST 5453; 1:500), Bcl-xL (CST 2764; 1:500), SDHA (Abcam, ab123545; 1:500), OXPHOS (Abcam, ab110411;1:500), ClpP (B-12) (Santa Cruz Biotechnology Inc, sc-271284; 1:500), ClpX (Abcam, ab203694, 1:500), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Thermo Fisher, 31460) and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Thermo Fisher, 31430). The antibodies were used in the protein capillary electrophoresis: HDAC1 (CST 34589; 1:25), HDAC2 (CST 57156; 1:25), and Vinculin (Abcam ab129002, 1:500).
Nguyen T.T., Shang E., Schiffgens S., Torrini C., Shu C., Akman H.O., Prabhu V.V., Allen J.E., Westhoff M.A., Karpel-Massler G, & Siegelin M.D. (2022). Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma. Clinical Cancer Research, 28(9), 1881-1895.
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7

Silver Staining of Protein Gels

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5μl of samples containing LDS buffer and DTT prepared for tandem affinity purification and mass spectrometry described above were mixed with 0.5 μl of 500 mM iodoacetamide (0210035105, MP Biomedicals). Proteins were separated in a 4–12% Bis-Tris gel (NP0321BOX, Invitrogen), followed by fixation of the gel overnight in 50% methanol at room temperature. The gel was impregnate with solution C (0.8% (w/v) silver nitrate (S6506, SIGMA), 207.2mM ammonium hydroxide (A6899, SIGMA) and 18.9 mM sodium hydroxide) for 15 minutes, followed by rinsing with water twice. The image was then developed in solution D (0.05% citric and 0.0185% formaldehyde in Milli-Q) until intensity of the bands increase to optimal level. The reaction was then terminated by adding stop solution (45% methanol and 10% acetic acid).
Kanie T., Ng R., Abbott K.L., Pongs O, & Jackson P.K. (2023). Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages. bioRxiv.
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8

Serum BDNF Protein Detection

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Western blots were performed as described53 . Proteins from 1 µl serum were separated on 4–12% Bis-Tris gradient gels (Invitrogen, NP0321BOX) and transferred to nitrocellulose membranes. After blocking for 1 h in 3% BSA (Sigma-Aldrich, A7906), 3% ECL Prime Blocking reagent (GE Healthcare) in TBS-T, membranes were incubated overnight with anti-BDNF 3C11 antibody (1:2000, Icosagen, 327-100). After washing, 1 h incubation with HRP-conjugated goat anti-mouse IgG1 (1:2000, Invitrogen, PA1-74421) followed, and membranes washed and developed.
Dingsdale H., Nan X., Garay S.M., Mueller A., Sumption L.A., Chacón-Fernández P., Martinez-Garay I., Ghevaert C., Barde Y.A, & John R.M. (2021). The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor. Translational Psychiatry, 11, 62.
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9

AZD-8055 Induced Protein Analysis

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Tissue was collected 1 h after vehicle or AZD-8055 administration (20 mg/kg or 100 mg/kg AZD-8055). The right frontal brain lobe was lysed from male mice that were 12 weeks old. Tissue was lysed in 1 mL cell extraction buffer (Invitrogen #FNN10011) supplemented with protease (Sigma #P7626) and phosphatase inhibitors (Sigma#P5726, #P0044) with a Dounce homogenizer. Lysate was centrifuged and the supernatant was collected for total protein quantification. Total protein (30 μg) was loaded to a NuPAGE 4-12 % Bis-Tris gel and subject to gel electrophoresis according to the manufacturer’s instructions (Invitrogen #NP0321BOX). Bands were detected by fluorescent imaging using the Typhoon imaging system.
Hornstein N., Torres D., Das Sharma S., Tang G., Canoll P, & Sims P.A. (2016). Ligation-free ribosome profiling of cell type-specific translation in the brain. Genome Biology, 17, 149.
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10

Western Blot Analysis of Apoptosis Markers

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Cells were lysed in the laemmli buffer (Biorad) containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA, 78440). Cell lysates were run on a 4–12% SDS PAGE gel (Invitrogen NP0321BOX), the proteins were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk (VWR (Bridgeport, NJ, USA) 97063-958) in TBST (0.1% Tween20) and probed with target antibodies. Primary antibody incubations were performed overnight at 4 °C. Antibodies were used Rpb1 (CST 14958), p-Rpb1 (CST 4735), PARP (CST 9532; 1:500), cCP9 (CST 7237; 1:500), cCP3 (CST 9665; 1:500), Mcl-1 (CST 5453; 1:500), Bcl-xL (CST 2764; 1:500), Bcl-2 (CST 4223; 1:500), Noxa (Calbiochem (Burlington, MA, USA) OP180, clone 114C307; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2000). The HRP linked secondary antibodies were from Santa Cruz Biotechnology Inc. The target protein was detected on the Azure (C300) imaging system. In selected cases, the protein capillary electrophoresis (Protein Simple (San Jose, CA, USA) SM-W004) was used. The following antibodies were applied Mcl-1 (CST 5453; 1:25), Bcl-xL (CST 2764; 1:25), Bcl-2 (R&D System, MN, USA, MAB827; 1:25), Noxa (Calbiochem OP180, clone 114C307; 1:25), and Vinculin (Abcam (Cambridge, MA, USA) ab129002, 1:500).
Shang E., Nguyen T.T., Shu C., Westhoff M.A., Karpel-Massler G, & Siegelin M.D. (2020). Epigenetic Targeting of Mcl-1 Is Synthetically Lethal with Bcl-xL/Bcl-2 Inhibition in Model Systems of Glioblastoma. Cancers, 12(8), 2137.
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