After quantification by the Bradford assay, equal amounts of total protein were boiled in NuPAGE SDS sample buffer (Thermo) containing a reducing agent. Three micrograms of whole-cell lysates and 10 ng of unprocessed enzymes were resolved on a 4 to 12% Bis-Tris Midi Gel (Thermo). Proteins were transferred to a nitrocellulose membrane using the iBlot 2 dry blotting system (Thermo). Membranes were blocked with 3% bovine serum albumin (BSA) in PBS with 0.1% Tween 20 for 30 min at room temperature (RT). After incubation, blots were probed with rabbit anti-human GAA (1:1000, 1 h, RT; ab137068, Abcam) or rabbit anti-actin (1:2500, 1 h, RT; ab179467, Abcam) and then probed with donkey anti-rabbit secondary antibody conjugated with IRDye 680 or 800 (1:5000–1:10,000, 1 h, RT; LI-COR) for detection. Odyssey CLx (LI-COR) was used for scanning blots. For Eastern/lectin blots, 0.5 to 1 μg of purified proteins were resolved on a 4 to 12% Bis-Tris mini gel (Thermo) and blotted as noted above. Membranes were blocked in 5% BSA in TBS-0.1% Tween 20 supplemented with 1 mM MnCl2 and CaCl2 for at least 1 h and incubated with 100 μg of biotinylated WGA (Vector Laboratories) diluted in the blocking buffer overnight at 4 °C. Blots were probed with streptavidin conjugated to IRDye 800 (1:10,000, 1 h, RT; LI-COR) and also probed for GAA as noted above.
Nupage sds sample buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE SDS sample buffer is a ready-to-use solution designed for preparing protein samples for electrophoresis analysis. It contains the necessary components to denature and solubilize proteins, allowing for their effective separation and detection.
Automatically generated - may contain errors
Lab products found in correlation
Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Odyssey clx, by LI COR (1 mentions)
Irdye 800, by LI COR (1 mentions)
Biotinylated wga, by Vector Laboratories (1 mentions)
Ab179467, by Abcam (1 mentions)
Ab137068, by Abcam (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Superblock buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Na3vo4, by New England Biolabs (1 mentions)
Genegnome xrq, by Syngene (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Hrp conjugated species specific secondary antibodies, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
13 protocols using nupage sds sample buffer
1
Western and Lectin Blotting of Glycosidase
2
Protein Quantification and Validation
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All individual samples were quantified for protein concentration using a BCA assay and following manufacturer’s instructions (Pierce™ BCA Protein Assay Kit, ThermoFisher Scientific). To check for accurate quantification and sample integrity, for every sample, 5 μg of protein mixed with NuPAGE™ SDS sample buffer (4×, ThermoFisher) was loaded into the wells of an NuPAGE 4–12% Tris-Bis gel. After electrophoresis, the gels stained overnight at room temperature with InstantBlue™ total protein stain. The gels were then imaged using an infrared scanner (Odyssey LI-COR biosciences, US) at 700 nm and analyzed using ImageStudio™ Lite (LI-COR). After subtraction of background signal, fluorescence intensity was measured for each sample on the totality of the lane and on three arbitrarily chosen regions (198–62, 62–28 and 28–6 kDa) (45 (link)).
3
HUVEC Cell Lysis and Western Blotting
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HUVEC cultures were lysed using RIPA buffer (Boston Bioproducts) supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and PhosStop Phosphatase Inhibitor Cocktail (Roche), 1 mM Na3VO4 (New England Biolabs), and 1 mM NaF. Proteins were resolved via SDS-PAGE 4%–12% gradient gels (Thermo Fisher Scientific) under reducing conditions using NuPAGE SDS sample buffer and sample reducing agent (Thermo Fisher Scientific), transferred to a nitrocellulose membrane, and blocked with SuperBlock buffer (Thermo Fisher Scientific). Protein detection was performed with the following primary antibodies: TMEM16E/Ano5 (clone N421A/85, UC Davis/NIH NeuroMab), V5-Tag (80076, Cell Signaling Technology), TMEM16F (MilliporeSigma), TFPI (AF2974, R&D Systems), β-actin, and GAPDH (12620 and 2118, respectively, Cell Signaling Technologies). Appropriate species-specific HRP-conjugated secondary antibodies were also used (Cell Signaling Technologies). Immunoblots were developed with Supersignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized with a GeneGnome XRQ (Syngene) or a ChemiDoc (Bio-Rad) and analyzed using ImageJ software (NIH).
4
Enzymatic Deglycosylation of rhGAA
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Bacterial neuraminidase from Arthrobacter ureafaciens recombinantly expressed in Escherichia coli (5 units/ml) was obtained from Sigma. 20 μg of each rhGAA sample was treated with 5 μl of a 1:100 diluted (in TBS) solution of bacterial neuraminidase for 20 h at 37 °C. The final pH of the reactions was adjusted to 4.8 with addition of 0.1 M sodium acetate (pH 4.8), and final reaction volumes were 40 μl. Untreated samples were subjected to the same conditions, except TBS was added instead of an exoglycosidase. 2.5 μg of treated rhGAA was denatured using NuPAGE SDS sample buffer (Thermo) containing a reducing agent and set aside for Eastern/Western blotting, while the remainder of the samples (containing 17.5 μg rhGAA) were flash-frozen and stored at −80 °C before denaturing with urea for MS analysis. NEU1 and NEU4 treatments were performed on 2 μg rhGAA by adding 10 μl of NEU1/NEU4 (concentrations of both were ∼2 mg/ml) or 2 μl 1:100 diluted (in TBS) bacterial neuraminidase in a final volume of 40 μl and a pH of 4.8, obtained by adding a final concentration of 0.1 M sodium acetate (pH 4.8) to the reaction mixture.
5
Western Blot Protein Analysis Protocol
6
Western Blot Analysis of HIV-1 Viral Proteins
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Cells were lysed in radioimmunoprecipitation assay buffer (RIPA, (25 mM Tris (pH 8.0), 137 mM NaCl, 1% glycerol, 0.1% SDS, 0.5% Na-deoxycholat, 1% Nonidet P-40, 2 mM EDTA, and protease inhibitor cocktail set III [Calbiochem, Darmstadt, Germany].) buffer, 20 min on ice. Lysates were clarified by centrifugation (10 min, 300 g, 4°C). Samples were boiled in NuPAGE SDS Sample Buffer and NuPAGE Sample Reducing Agent (Life technologies) and subjected to SDS-PAGE followed by transfer to a PVDF membrane. A3G and A4 Proteins were detected using an anti-HA antibody (Ab) (1:104 dilution, MMS-101P; Covance, BioLegend, Fell, Germany), HIV-1 p24 Gag was detected applying HIV-1 p24 monoclonal Ab (1:250 dilution, AG3.0, NIH AIDS REAGENTS, Germantown, USA) [91 (link)]. Cell lysates were probed with α-tubulin Ab (1:104 dilution, B5-1-2; Sigma-Aldrich, Munich, Germany) and virions with α-p24 monoclonal Ab 183-H12-5C. Vif protein was detected with HIV-1 Vif monoclonal antibody (1:5x103 dilution, #319, NIH AIDS REAGENTS) [92 (link)]. Secondary Abs.: anti-mouse (NA931V) and anti-rabbit (NA934V) horseradish peroxidase (1:104 dilution, GE Healthcare). Signals were visualized using ECL reagent (GE Healthcare).
7
TRIM60 Interactome Profiling by IP-MS
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RAW cells overexpressing HA-TRIM60 were stimulated with LPS for 30 min. TRIM60-interacting proteins were purified by immunoprecipitation using anti-HA magnetic beads (88836, Pierce). The IP products were subjected to mass spectrometric analysis. Briefly, the washed IP beads were boiled in 30 μl of 1X NUPAGE SDS sample buffer (Invitrogen) and then separated by SDS-PAGE (NuPAGE 10% Bis-Tris Gel, Invitrogen). The proteins in the gel were visualized by Coomassie Brilliant blue staining and divided into several gel pieces according to molecular weight. The individual gel pieces were destained, and proteins were subjected to in-gel digestion with trypsin (Promega). The tryptic peptides were eluted, lyophilized, resuspended in 10 μl of loading solution (5% methanol containing 0.1% formic acid) and analyzed by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a nano-LC 1000 system (Thermo Fisher Scientific) coupled to a Q Exactive plus (Thermo Fisher Scientific) mass spectrometer. The raw MS data were analyzed by Mascot.
8
Protein Expression Profiling by Western Blot
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The samples were diluted in NuPAGE SDS Sample buffer (Invitrogen NP0007) and the SDS-PAGE was performed using NuPAGE Novex Bis-Tris Mini Gels 4–12 %. SDS-PAGE gels were electrobloted in iBlot Device and the membranes were incubated with primary antibodies HSPB1/HSP27 and HSP90B1(GRP94) from Cell Signaling Technology; NPM and RKIP from Zymed-Invitrogen; NCL and β-actin from Santa Cruz Biotechnology; NOVA-1 from Sigma-Aldrich. The same source of antibodies HSPB1 and NOVA1 were used for immunohistochemistry.
9
LRP1 and PID1 Interaction in BAT and Muscle
10
Immunoprecipitation Protocol for Protein Analysis
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For immunoprecipitation, 1 mg of cell lysate was prepared with 1% NP buffer (1% Igepal CA-630, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and protease inhibitor mixture). One microgram of primary antibody was added and allowed to probe for 2 h with continuous rotation at 4 °C. Ten microliters of protein A/G magnetic beads (LSKMAGAG02, Millipore, Burlington, MA, USA) were added to collect antibodies. After 2 h of continuous rotation at 4 °C, beads were precipitated with a magnet and washed twice with 1% NP buffer. The precipitants were denatured and reduced by boiling for 5 min after adding NuPAGE SDS sample buffer (Invitrogen, Waltham, MA, USA, NP0007) with beta-mercaptoethanol (Sigma, St. Louis, MO, USA, #63689). Proteins were separated by SDS-PAGE, and 30 μg of protein was utilized for input control.
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