Wild-type and mutant α subunits (780 μg) were loaded on to a HiPrep Sephacryl S-300 HR column (GE Healthcare) coupled to an AKTA Prime Plus chromatography system (GE Healthcare). Elution profiles were analyzed using Prime View evaluation software. The column was equilibrated with Buffer D (25 mM Tris-HCl, pH 7, 150 mM NaCl), the flow rate was 0.8 ml min−1, and 3 ml fractions were collected. Calibration of the column was carried out using 360 μg of each of six molecular weight standards (Serva). Aliquots (15 μl) of sizing column fractions were mixed with 5× SDS sample buffer and analyzed by 12% SDS-PAGE followed by staining with GelCode blue. In addition, aliquots (50 μl) of sizing column fractions were mixed with 5× nondenaturing sample buffer and analyzed by nondenaturing 4–15% gradient precast gels followed by staining with Imperial Stain (ThermoScientific) or Pierce Silver Stain Kit (ThermoScientific). In experiments requiring the pooling of sizing column fractions, the indicated fractions were combined and concentrated down to a volume of 0.6 ml using Pierce Protein Concentrators, 9K (ThermoScientific). These pooled and concentrated fractions were then mixed with crude lysates of BL21 cells expressing untagged archaeal β subunits. Proteins were repurified by ICAR and analyzed by native PAGE and substrate-overlay assay as described above.
Imperial stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Imperial Stain is a laboratory equipment product designed for staining microscopic specimens. It is a versatile staining solution used to enhance the visibility and contrast of cellular structures and tissues for analysis and examination purposes.
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Lab products found in correlation
Hiprep sephacryl s 300 hr column, by GE Healthcare (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Akta prime plus, by GE Healthcare (1 mentions)
M2 flag conjugated magnetic beads, by Merck Group (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Np0336box, by Thermo Fisher Scientific (1 mentions)
Bio safe coomassie g 250, by Bio-Rad (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Asp n protease, by Roche (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Reprosil pur c18 aq, by Dr. Maisch (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Mops buffered sds page gel, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Milli q water, by Merck Group (1 mentions)
12 protocols using imperial stain
1
Purification and Characterization of Archaeal α and β Subunits
2
Identification of Ago2-Associated Proteins
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TT-Ago2 (control), TT-FHAgo1, or TT-FHAgo2 cells were induced with Dox (2.5 µg/mL) and fractionated to cytosolic and nuclear fractions. Cytosolic and nuclear fractions were dialyzed in buffer DB (20 mM Tris-Cl pH 8, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA) for 6 h with four buffer changes. Dialyzed lysates were centrifuged and Flag IP was carried out overnight with M2 Flag conjugated magnetic beads (Sigma). Immunoprecipitated material was washed and eluted with 3× Flag peptide (Sigma) twice at room temperature. The eluted material was TCA precipitated overnight at 4°C. The precipitate was resuspended in gel loading buffer, separated utilizing 4%–12% SDS-PAGE (Thermo Fisher Scientific), and visualized with Imperial stain (Thermo Fisher Scientific). The gel was separated into eight equal size pieces and submitted to the Biopolymers & Proteomics Core Facility of Robert A. Swanson (1969) Biotechnology Center at the Koch Institute for mass spectrometry.
3
Enzymatic Digestion of PARP Proteins
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Trypsin was added at a ratio of 1:2400 (w/w) to 2.4 μg of NTR (Figure 2B , top) or FL PARP-2 (Figure 2C ). Trypsin was added at a ratio of 1:500 (w/w) to 2.4 μg of PARP-1 Zn1 and PARP-2 NTR (Figure 2B , bottom). Reactions were incubated at room temperature for the indicated time points. Reactions were quenched by the addition of SDS-PAGE loading dye and then boiled for 5 min at 95°C. Proteolytic products were resolved on 18% (NTR) or 7.5% (FL PARP-2) SDS-PAGE and the gels were treated with Imperial Stain (Thermo scientific) for visualization or used for western blot analysis. A representative image of three independent experiments is shown.
4
SDS-PAGE Fractionation and Visualization
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For each growth condition (MHB, BHI, or BCA), the WC and 4 subfractions (A, D, SS, SI) were run on SDS-PAGE gels either Full-Length (WC and A), or in a Gel Plug (D, SS, SI). Each fraction was mixed with sample buffer (w/β-ME) to a final vol of 100 μl. Full-Length: loaded 10 μl (1 × 108Ft eq)/lane on 4–12% SDS-PAGE gradient gels ran at 50 V for 4 h (Invitrogen). Gel-Plug: loaded 10 μl (5 × 108Ft eq)/lane on 12% SDS-PAGE gel ran at 50 V for 40 min to create “1D-Gel plug” (Invitrogen). All SDS-PAGE gels were stained with 25 mL Imperial Stain (Thermo 24615) for 1 h at 22°C and destained overnight at 4°C.
5
Protein Separation and Identification from Whole Cell Lysates
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For the whole cell lysate samples, 20 μg of whole cell extract from KG-1 cells (determined by Bradford assay, Thermo #1856209 using λ595 nm) was loaded onto a 4–12% MOPS buffered 1D SDS-PAGE gel (Invitrogen NP0336BOX) and run at ~ 200 V for ~ 45 min. For the IP samples, 30% of the IP eluate (30 μL/100 μL) was loaded. The gel was stained with Imperial Stain (Thermo #24615) for 1 h at room temperature. For the whole cell lysate samples, the “p200” sections were excised from the gel by sterile razorblade (MW range 150–225 kDa) and the “p75” sections were excised in the MW range of ~ 60–75 kDa and in-gel trypsin digested as described below. For the IP samples, gel sections were excised as follows: “p200” sections MW range (~ 150–250 kDa) and “p75” sections MW range (~ 65–90 kDa) and in-gel trypsin digested as described below.
6
Isolation and Analysis of Mitochondrial Respiratory Supercomplexes
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Mitochondrial fractions were isolated from fresh calf muscle by the Potter–Elvehjem method. Purified mitochondria were solubilized in digitonin according to the procedure69 (link) with minor modifications. To extract the respiratory SCs, mitochondria purified from the muscle tissue were solubilized by mild detergent digitonin (8 g/g of protein)7 (link). After digitonin extraction, the respiratory chain complexes and SCs were separated by BN-PAGE and subjected to in-gel enzyme activity staining or western blot analysis with antibodies against subunits of Complexes I, III, and IV. Positions of Complex V (F0F1 ATPase) and its dimer (V2) were also identified by in-gel enzyme activity staining41 (link). Mitochondrial lysates were subjected to BN-PAGE in a 3–12% acrylamide gradient gel in Bis-Tris buffer (Invitrogen) according to manufacturer’s instructions. After electrophoresis, gels were either stained with Imperial stain (Thermo Fisher Scientific) and Bio-Safe Coomassie G-250 (Bio-Rad) or used for western blotting analysis. For CI and CIV in-gel activity staining, mitochondrial lysates were subjected to BN-PAGE41 (link). See Supplementary Methods for additional details.
7
SDS-PAGE Analysis of Lysozyme Variants
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SDS-PAGE analyses (reported in the Supplementary Materials ) were performed using an Invitrogen Mini Gel Tank instrument and a Novex Wedge gel 4 to 20% Tris-Glycine, 1.0 mm, Mini Protein Gel 10Well by seeding the following volumes of sample solutions (from left to right):
LMW calibration kit for SDS electrophoresis (from 14.4 to 97.0 Dalton): 5 µL
Lysozyme HCl: 8 µg
LysOHT®: 8 µg
Lysozyme HCl heat treated in aqueous solution (0.1% aqueous solution for 20′ at 100 °C): 8 µg
8
Mass Spectrometry Analysis of GFP-LC3A
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GFP-LC3A samples were analyzed by mass spectrometry as described in Durgan et al. (2021) (link). In brief, samples were run on 10% NuPAGE gels in MOPS buffer (Invitrogen). Each gel was washed and stained with Imperial Stain (24615; Thermo Fisher Scientific) for 2 h and then destained in dH2O overnight. The gel region containing GFP-LC3A was excised into a single tube, destained, and typically saponified by treatment with 50 mM NaOH in 30% MeOH at 40°C for 2 h. The protein was digested with AspN protease (Roche) at 30°C for 16 h, in 25 mM ammonium bicarbonate. For targeted mass spectrometric assay of modified C-terminal LC3A peptides, samples were separated on a reversed-phase nanoLC column (150 × 0.075 mm; Reprosil-Pur C18AQ, Dr. Maisch), interfaced to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) operating in high resolution (orbitrap) MS1 mode, with the data-dependent acquisition of low-resolution MS2 spectra generated by CID in the linear ion-trap. Quantitative data were extracted using Skyline software (MacCoss Lab, University of Washington) using the sum of the chromatographic peak areas from the y1 to y10 fragment ions. Subsequently, normalization was performed against the unmodified C-terminal peptide.
9
In-Gel Digestion of EV Samples
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For the in-gel digestion, 20 µg of EV samples obtained by the large-scale PEG method were separated by SDS PAGE. Staining was obtained by Imperial Stain according to the manufacturer’s instructions (Thermo Fisher). After electrophoresis gels were washed three times for 5 min with distilled water and stained for 2 h. Subsequently, each gel lane was cut in five equal pieces. Destaining, alkylation, reduction, tryptic digestion and peptide extraction was performed according to Schrotter and colleagues [46 ].
10
SUMO-SAM Protein Purification
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