Np0004

Manufactured by Thermo Fisher Scientific

The NP0004 is a general-purpose laboratory instrument designed for a variety of applications. It features basic functionality for sample preparation, analysis, or processing tasks. The device specifications and capabilities are suitable for a wide range of laboratory workflows.

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11 protocols using np0004

Antibody-Antigen Complex Purification

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An antibody-antigen complex was prepared by adding 100 µg of protein lysate and primary antibody in RIPA buffer to a total volume of 1 ml, then incubated overnight at 4 °C on a rotary tube mixer. The following day, 50 µl of protein-G magnetic beads (Millipore, LSKMAGG10) were dispensed into a 1.5 ml collection tube and placed on a magnetic rack (Millipore, 20–400). The storage substrate was removed and the beads were washed twice in 500 µl PBS with 0.1% Tween-20 (PBST). The mixture was briefly vortexed and centrifuged between washes and the supernatant was discarded. Next, the antibody-antigen complex was mixed with the magnetic beads and incubated overnight on a rotary tube mixer at 4 °C. The next day, the mixture was briefly centrifuged and washed 3 times in PBST. The elution was resuspended in 25 µl RIPA buffer with reducing agent (ThermoFisher, NP0004) and sample buffer (ThermoFisher, NP0007). Next, the samples were denatured by heating at 95 °C for 10 and loaded into 4–12% Bis-Tris pre-cast protein gels for SDS-PAGE.

Alshehri B., Pagnin M., Lee J.Y., Petratos S, & Richardson S.J. (2020). The Role of Transthyretin in Oligodendrocyte Development. Scientific Reports, 10, 4189.

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SDS-PAGE Protein Denaturation and Separation

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A volume containing 5 µg of protein, 1 x sample buffer (ThermoFisher, NP0007), 1 x reducing agent (ThermoFisher, NP0004) and ddH2O were denatured by heating at 95 °C for 5 minutes (or 100 °C for 15 minutes for TTR monomer detection). The sample mixture was cooled on ice for 5 minutes before being briefly centrifuged. Samples and protein ladder (ThermoFisher, 26634) were loaded into 4–12% Bis-Tris pre-cast protein gels (ThermoFisher, NP0335) or 12% Bis-Tris pre-cast protein gels for TTR controls (ThermoFisher, NP0341), in MOPS running buffer supplemented with 1:400 antioxidant (ThermoFisher, NP0005). Electrophoresis (Biorad, Mini-protean) was set to 150 V for 1.5 hours at room temperature.

Alshehri B., Pagnin M., Lee J.Y., Petratos S, & Richardson S.J. (2020). The Role of Transthyretin in Oligodendrocyte Development. Scientific Reports, 10, 4189.

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Proteomic Analysis of NL4-3 Viral Lysates

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The lysate of concentrated NL4-3 viruses treated with DMSO or Indinavir (~13 μl) was mixed with 5 μl 4× LDS sample buffer (ThermoFisher, NP0007) and 2 μl 10× sample reducing agent (ThermoFisher, NP0004). The entire mixture (20 μl) was heated at 95°C for 5 minutes and loaded on a 4–12% Bis-Tris denaturing protein gel, together with protein molecular weight standards (Bio-Rad, 1610374). The gel was stained with Pierce silver stain kit (ThermoFisher, 24612) by following the manufacturer’s instructions. Protein bands around the target fragment band region were excised from the gel using the protein molecular weight standard lane as a guide. The gel bands were sent to the Proteomics & Metabolomics Facility at the Center for Biotechnology/University of Nebraska–Lincoln for protein identification mass spectrometry. Mascot (Matrix Science, London, UK; version 2.6.1) was used to analyze all MS/MS samples. MS/MS based peptide and protein identifications were further analyzed by Scaffold (version Scaffold_4.8.4, Proteome Software Inc., Portland, OR).

Jurczyszak D., Zhang W., Terry S.N., Kehrer T., Bermúdez González M.C., McGregor E., Mulder L.C., Eckwahl M.J., Pan T, & Simon V. (2020). HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle. PLoS Pathogens, 16(2), e1008305.

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MAGL Activity Quantification in Mouse and Human Brain

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Homogenates of whole mouse brains or human cortical brain tissue (BioIVT, Westbury, NY, USA) without disease pathology were prepared in ABPP lysis buffer. Informed consent agreements were obtained from all donors according to BioIVT policies. Tissue in ABPP lysis buffer was homogenized as described above, and the lysate supernatants were retained. We incubated 50 μg of the lysates with 0.5 μL DMSO, 10 μM MAGLi 432 or 10 μM JZL 184 for 25 min, and then with 0.5 μL of 200 nM TAMRA-FP (green) or 5 μM MAGL-specific fluorescent probe [40 ] for 30 min, protected from light. The reaction was stopped by adding 12.5 μL deionized water, 12.5 μL 4x sample buffer (Thermo Fisher Scientific; NP0007) and 5 μL reducing agent (Thermo Fisher Scientific; NP0004). We loaded 25 μL of cell lysate solution into the wells of NuPAGE 10% Bis-Tris gels (Invitrogen; NP0301BOX) and run for 55 min at 200 V. We measured in-gel fluorescence on the ChemiDoc MP System (Bio-Rad) using Cy3 and Cy5 filters. The membrane was then stained with Coomassie Brilliant Blue for total protein quantification (S1 Fig).

Kemble A.M., Hornsperger B., Ruf I., Richter H., Benz J., Kuhn B., Heer D., Wittwer M., Engelhardt B., Grether U, & Collin L. (2022). A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature. PLoS ONE, 17(9), e0268590.

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Western Blot Analysis of KCa3.1 in Brain Microvessels

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Bovine CMEC whole cell lysates from cells grown for 48 h in ACM containing medium and freshly isolated rat brain microvessel lysates were subjected to Western blot analysis. Rat brain microvessels were isolated as described.^{25 (link)} Five mg protein from each sample was denatured with 50 mM dithiothreitol (DTT) in SDS sample reducing buffer (NP0004, Life Technologies) and heated at 70°C for 10 min. Protein samples were then separated on 12% Tris-HCl gel (No.5952, PAGEr™ Gold Precast Gel, Lonza) using electrophoresis and electrotransferred to polyvinylidene fluoride membranes (BioRad XCell SureLock, Hercules). Membranes were blocked with 7.5% milk in PBS with 0.1% Tween-20 (PBST) for 1hr, incubated with anti-KCa3.1 antibody (AV35098, rabbit polyclonal, 1:3000, Sigma-Aldrich, Louis, MO) in milk/PBST overnight at 4°C, washed with PBST, and incubated with horseradish peroxidase-conjugated secondary antibody (A16096, goat anti-rabbit, 1:2000, Life Technologies) in milk/PBST for 1 hr at room temperature. After washing with PBST, bound antibody was detected using enhanced chemiluminescence kit (RPN2133, ECL plus; GE Healthcare) and visualized on a Fuji Film LAS-4000 Imaging Machine (Medford, UK).

Chen Y.J., Wallace B.K., Yuen N., Jenkins D.P., Wulff H, & O’Donnell M.E. (2014). Blood Brain Barrier KCa3.1 Channels: Evidence for a Role in Brain Na Uptake and Edema in Ischemic Stroke. Stroke; a journal of cerebral circulation, 46(1), 237-244.

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Immunoprecipitation of HSF1 Protein

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Tissues were dounce homogenized in ice-cold PBS supplemen-ted with complete protease inhibitors (11697498001; Roche) and phosphatase inhibitors [1 mm sodium orthovanadate (P0758S; New England Biolabs), 50 mm NaF (201154; Sigma), 10 nm okadaic acid (08010; Sigma)]. Lysates were used immediately and never frozen. Samples were spun at 13 000 g at 4°C for 15 min, and the supernatants transferred to a fresh tube. Protein concentrations were determined using a BCA-protein assay (23 223 and 23 224; Thermo Scientific). 0.2–0.5 mg protein lysate was incubated with 3 µg anti-HSF1 antibody (SPA-901; Stressgen) or normal rabbit IgG (#2729; Cell Signaling) for 4 h at 4°C on a rotating wheel (SB3 rotating wheel; Stuart Instruments). Protein G-coupled Dynabeads (10004D; Life Technologies) were added to each IP and incubated at 4°C for 45 min on a rotating wheel. Following immunoprecipitation, the protein G-coupled Dynabeads were briefly spun and put on a magnetic rack, washed in 4% Triton X-100/PBS (3×) and finally resuspended in 4× loading buffer (NP0007; Life Technologies) with a reducing agent (NP0004; Life Technologies). Immunoprecipitated complexes were eluted by denaturation at 95°C for 10 min and run on a 4–12% precasted gel (WG1402BOX; Life Technologies) according to the manufacturer's instructions.

Carnemolla A., Labbadia J.P., Lazell H., Neueder A., Moussaoui S, & Bates G.P. (2014). Contesting the dogma of an age-related heat shock response impairment: implications for cardiac-specific age-related disorders. Human Molecular Genetics, 23(14), 3641-3656.

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Comparative Proteomics of Lamprey Spinal Cord Injury

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The SCs of lamprey were collected from a control animal and one at 2
weeks post transection. A mouse brain was included as a control. The tissues
were snap-frozen by liquid nitrogen and homogenized by sonication in cold lysis
buffer (C3228, Sigma-Aldrich) supplemented with 1X protease inhibitor cocktail
(P8341, Sigma-Aldrich). After brief centrifugation to remove debris, the total
protein concentration in supernatants was determined by using Bio-Rad (Hercules,
CA) DC protein assay reagents. After 5 minutes of heating at 75°C in
loading buffer (NP 0007, Invitrogen) supplemented with reducing reagent (NP
0004, Invitrogen), 30 or 25 μg of SC proteins from each lamprey and 50
μg mouse brain proteins were loaded. Protein was separated in 3–8%
NuPAGE® Tris-acetate gradient mini gels (EA0375BOX, Invitrogen). The
protein was transferred onto PVDF membrane by using a Bio-Rad transblot
apparatus. The membranes were blocked in 5% nonfat dry milk in TBS buffer for 1
hour at RT and probed with different CSPG antibodies (CS-56 or Cat-316) at
4°C overnight. After washes with TBS, the blots were incubated with
secondary antibody goat anti-mouse IgM conjugated with Alexa Fluor 488 for 2
hours at RT, scanned with a Typhoon 9400 imager (GE Healthcare Biosciences,
Piscataway, NJ), and processed in Adobe (San Jose, CA) Photoshop to adjust
contrast and brightness.

Zhang G., Hu J., Li S., Huang L, & Selzer M.E. (2014). Selective Expression of CSPG Receptors PTPσ and LAR in Poorly Regenerating Reticulospinal Neurons of Lamprey. The Journal of comparative neurology, 522(9), 2209-2229.

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Western Blot Analysis of Sf9 Cells

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One million of transfected Sf9 cells were pelleted and the supernatant was removed. Pellets were resuspended in 100 μL of lysis buffer (Tris/phosphate 25 mM, pH 7.8, Glycerol 15%, DTT 1 mM, EDTA 1 mM, MgCl₂ 8 mM, Triton 0.2%) with Protease Inhibitor Cocktail (04693116001—Roche). Samples were kept on ice for 30 minutes. 20 μL was mixed with LDS 4X (NP0007—Invitrogen) and 10X reducing agent (NP0004—Invitrogen). Samples were then heated 30 minutes at 94°C and ran on a polyacrylamide gel (NuPAGE 4–12% Bis-Tris Gel—Invitrogen). After migration, proteins were transferred onto a nitrocellulose membrane using the iBlot transfer system (Invitrogen, following manufacturer protocol). Membranes were saturated for 1h with blocking buffer (Odyssey). Rep detection was performed with Ig 259.5 (65158—Progen) whereas eGFP detection was performed using ab1218 (9F9.F9—Abcam) as primary antibody. Both were diluted 1/250. Antibody 680 LT (925–68020 Li-cor) diluted 1/20.000 was used as secondary Ab. WB against baculoviral p35 protein was performed with NB100-92383 rabbit antibody (Novus biologicals) diluted 1/5.000 as primary Ab and Ab 800 CW diluted 1/20.000 (925–32211 Li-cor) as secondary Ab. Images were acquired with Odyssey imager (Li-cor).

Savy A., Kaikkonen M.U., Léger A., Dickx Y., Galibert L, & Merten O.W. (2018). Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system. PLoS ONE, 13(7), e0199866.

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Western Blot Analysis of Sf9 Cell Lysates

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Sf9 transfected cells were pelleted, and the supernatant was removed. Pellets were re-suspended in 100 μL of lysis buffer (Tris/phosphate 25 mM, pH 7.8, Glycerol 15%, DTT 1 mM, EDTA 1 mM, MgCl₂ 8 mM, Triton 0.2%) with Protease inhibitor cocktail (04693116001; Roche). Samples were kept on ice for 30 min before being centrifuged at 10,000 rpm for 10 min. The supernatant (20 μL) was mixed with LDS 4 × (NP0007; Invitrogen) and 10 × reducing agent (NP0004; Invitrogen). Samples were then heated for 15 min at 94°C and were run on a polyacrylamide gel (NuPAGE 4–12% Bis-Tris Gel; Invitrogen). After migration, proteins were transferred onto a nitrocellulose membrane using the iBlot transfer system (Invitrogen), following the manufacturer's protocol. Membranes were incubated for 1 h with blocking buffer (Odyssey). Membrane blotting was made with first antibody immunoglobulin B1 at 1/250 dilution (65158; Progen) and secondary antibody 680 LT (925-68020; Li-cor) diluted 1/20,000, following the manufacturer's instructions, and the image was acquired with an Odyssey device (Li-cor).

Savy A., Dickx Y., Nauwynck L., Bonnin D., Merten O.W, & Galibert L. (2017). Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System. Human Gene Therapy Methods, 28(5), 277-289.

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Aspirin Modulates BMAL1 Acetylation

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MOVAS (a mouse aorta smooth muscle cell line) and SVEC4-10 cells (a mouse vascular endothelial cell line) were obtained from American Type Culture Collection (CRL-2797 and −2181, respectively). Primary culture of mouse aorta smooth muscle cells (VSMC) was described previously²¹ (link). At 80% confluence, the cells, grown in a 12-well plate, were treated with 1mM aspirin for 24 hours, and then lysed in cell extraction buffer (Cat. No. FNN0011, Invitrogen). The cell lysates were applied for immunoprecipitation (IP) using rabbit antibodies (bmal1, sc-48790, Santa Cruz; Ac-Lysine, 9814, Cell Signaling Technology) and Dynabeads Protein G (100.03D, Invitrogen) following manufacturer’s instruction. The precipitated proteins were eluted using NuPAGE LDS sample buffer (NP0007, Invitrogen) containing a reducing agent (NP0004, Invitrogen) at 70℃. The eluted samples were then resolved by SDS-PAGE gel, transferred to PVDF membranes (LC2002, Invitrogen) and analyzed by using antibodies against bmal1, Ac-lysine, and p53 (sc-126, Santa Cruz). As negative controls, IP was performed in the absence of antibody or in the presence of rabbit IgG.

Chen L., Yang G., Zhang J., Ren B., Tang S., Li X, & FitzGerald G.A. (2018). Time dependent hypotensive effect of aspirin in mice. Arteriosclerosis, thrombosis, and vascular biology, 38(12), 2819-2826.

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