19 μl purified VPS13D LTD (1.5 mg/ml) was mixed with 1 μl of either NBD-labeled PA, PC, PE, PS, ceramide, C12 (1 mg/ml in methanol) or BODIPY-C16 (1 mg/ml in DMSO) in 20-μl total reaction volumes and incubated at 4 °C for 3 h. Samples were loaded onto 12% Precast Native gels with Hepes-Tris buffer and run for 4 h at 90 V on ice. NBD/BODIPY fluorescence was visualized using a Bio-rad ChemiDoc XRS + (170-8265). Then gels were stained with Coomassie blue G250 (20279; ThermoFisher Scientific) to visualize total proteins.
Coomassie blue g 250
Manufactured by Thermo Fisher Scientific
Sourced in United States
Coomassie Blue G-250 is a dye used for the colorimetric detection and quantification of proteins in solution. It binds to proteins, forming a colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Nativepage system, by Thermo Fisher Scientific (5 mentions)
Mitochondria extraction kit, by Solarbio (3 mentions)
Bicinchoninic acid (bca), by Solarbio (3 mentions)
Digitonin, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene di uoride membranes, by Thermo Fisher Scientific (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Zeba spin column, by Thermo Fisher Scientific (1 mentions)
4 methylumbelliferyl β d glucopyranoside mug, by Merck Group (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Antimycin a aa, by Merck Group (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Nativepage novex 4 16 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Light blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Dark blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
19 protocols using coomassie blue g 250
1
Lipid Binding Assay for VPS13D
2
Protein separation and enzymatic activity visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS–PAGE was performed with 8–16% Protean TGX protein gradient gels (Bio-Rad) with the Tris-glycine-SDS buffer57 (link). Blue Native (BN)–PAGE58 (link) was performed with 3–12% NativePAGE Bis-Tris protein gradient gels (Thermo Scientific) in presence of 0.02% Coomassie Blue G-250. For subunit analysis of native complexes, individual lanes from the BN–PAGE were excised, incubated in 2% SDS and 160 mM dithiothreitol (DTT), and denatured at 95 °C for 10 min, unless otherwise indictated (Fig. 3e ). Proteins were separated with 8% polyacrylamide gels, which were hand cast. Protein bands were stained with SimplyBlue SafeStain Coomassie Blue dye (Thermo Scientific) according to the manufacturer’s instructions.
Protein bands with activity on CMC and xylan were visualized using modification of the zymogram technique, as described previously15 (link). Gels were incubated in 2% wt/vol CMC or 2% wt/vol birchwood xylan solutions followed by incubation at 60 °C for up to 2 h in reaction buffer (25 mM MES, pH 6.0). In-gel enzymatic activities were visualized by incubating gels with a 0.5% Congo Red solution for 15 min and subsequent multiple washing steps with 20% NaCl.
Protein bands with activity on CMC and xylan were visualized using modification of the zymogram technique, as described previously15 (link). Gels were incubated in 2% wt/vol CMC or 2% wt/vol birchwood xylan solutions followed by incubation at 60 °C for up to 2 h in reaction buffer (25 mM MES, pH 6.0). In-gel enzymatic activities were visualized by incubating gels with a 0.5% Congo Red solution for 15 min and subsequent multiple washing steps with 20% NaCl.
3
Coomassie Blue Protein Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following dilution in 6X SDS protein loading buffer, 5 µg of OMVs (protein content), total protein extracts from E. coli MG1655 and fractions of intermediate stages (supernatant filtrate 0.45 or 0.22 µm) were charged on a 7.5% Mini-PROTEAN® TGX™ Precast Protein Gel (BIORAD, Cat No. 4561025) with the recommended running buffer (Tris-Glycine buffer). The gel was then soaked for 5 min with shaking in 0.5% Coomassie Blue G-250 (Thermofisher, Cat. No. 20279; prepared in 50% methanol, 10% acetic acid). The excess staining was removed with the destaining solution (40% methanol and 10% acetic acid) to allow visualization of proteins as blue bands on a clear background.
4
Isolation and Analysis of Mitochondrial Respiratory Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BN-PAGE experiments were performed on mitochondria isolated as described above. ETC complexes and supercomplexes were extracted at 4 °C for 2 min in the presence of 2% n-dodecyl-β-D-maltoside (DDM) and 2% digitonin, respectively, starting from 200 μg of mitochondria in a buffer composed of 1 M aminocaproic acid, 50 mM Bis Tris pH 7. After extraction mitochondria were spinned at 100,000 g for 30 min and supernatants were collected and loaded on polyacrylamide Native-PAGE 3–12% Bis-Tris gradient gels (Thermo Fisher Scientific) after addition of Coomassie Blue G250 (Thermo Fisher Scientific). Protein complexes were then visualized after 18 h of Coomassie Blue G-250 staining and/or subjected to in gel activity assay (see below). Bands corresponding to the indicated respiratory chain complexes were cut and subjected to 2D-SDS-PAGE, in order to separate single protein components, which were identified by Western immunoblotting.
5
SDS-PAGE Protein Separation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS PAGE was performed according to Laemmli [51 (link)]. Initially, 40 μL of the proteins extract were incubated with 8 μL sample buffer (0.106 M Tris-HCl buffer at pH 8.5, containing 0.51 mM of EDTA, 2% of lithium dodecyl sulfate, 10% of glycerol, 0.22 mM of Coomassie Blue G 250 and 0.175 mM of Phenol Red, Thermo Fischer Scientific, Waltham, E.U.A.) at 90 °C for 5 min. After that, 20 μL of the solution and 5 μL of the standard (Page RulerTM Prestained Protein Ladder (10–250 kDa), Thermo Fischer Scientific, Waltham, MA, USA) were separated by SDS-PAGE (NuPAGETM 12% Bis-Tris Gel, Thermo Fischer Scientific, Waltham, MA, USA) by loading the solution, followed by staining with Coomassie Brilliant blue R250 (in 10% acetic acid) and destaining with 10% acetic acid. After scanning the gels, the band intensity and the relative molecular weight of proteins were estimated using Image Lab software (version 6.0.1.34, Bio-Rad Laboratories, Hercules, CA, USA).
6
Desthiobiotinylation Protein Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Interactions between MAL and TRAM on cell lysates after infection were evaluated following previously described protocols35 (link)
,36
and using the kit containing the EZ-Link Sulfo-NHS-LC-Desthiobiotin enzyme according to manufacturer’s instructions (Thermo Fisher). Briefly, removal of excess desthiobiotin reagent from samples was performed using Zeba spin columns (Thermo Fisher). After coupling the desthiobiotinylated bait protein to the streptavidin resin, cell lysates were incubated for at least 1 h to finally elute the desthiobiotinylated bait protein and captured proteins. Obtained samples were then analyzed by SDS-PAGE and stained with Coomassie Blue G-250 (Thermo Fisher).
,36
and using the kit containing the EZ-Link Sulfo-NHS-LC-Desthiobiotin enzyme according to manufacturer’s instructions (Thermo Fisher). Briefly, removal of excess desthiobiotin reagent from samples was performed using Zeba spin columns (Thermo Fisher). After coupling the desthiobiotinylated bait protein to the streptavidin resin, cell lysates were incubated for at least 1 h to finally elute the desthiobiotinylated bait protein and captured proteins. Obtained samples were then analyzed by SDS-PAGE and stained with Coomassie Blue G-250 (Thermo Fisher).
7
Comparative Zymography of Recombinant Cellulases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zymography was performed to compare β-glucosidase and cellobiohydrolase activity of A5IL97 expressed in E. coli and A. niger. Unpurified recombinant proteins were not boiled and loaded with Novex® Tris-Glycine native sample buffer (2×; Thermo Fisher Scientific, Waltham, MA) into native PAGE gel without SDS. Following electrophoresis, the gels were incubated with 2.5% (v/v) of Triton-X for 1 hour, and then, further incubated with 50 mM sodium citrate, pH 5.0, containing 0.001% of 4-methylumbelliferyl-β-D-glucopyranoside (MUG; Sigma, St. Louis, MO) or 4-methylumbelliferyl-β-D-cellobioside (MUC; Sigma, St. Louis, MO) at 70°C for 10 minutes [25 (link)]. The gel was visualized under ultraviolet light then stained with Coomassie Blue G-250 using the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). The zymography assay was repeated at least two times.
8
Comprehensive 2D-PAGE Protein Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 2D-PAGE was done using a standard protocol [23 ]. The protein sample (1000 μg/mL) was purified using a 2-D gel clean up kit (GE Healthcare) according to the manufacturer’s instructions, and subsequently dissolved in 450 μL loading buffer (8 M urea, 2% w/v CHAPS, 0.5% v/v IPG buffer, 40 mM dithiothreitol (DDT), and 0.002% bromophenol blue). Isoelectric focusing (IEF) was performed at (30 V for 6 h, 60 V for 6 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 8000 V for 2.30 h, 8000 V for 7 h, and 500 V for 5–20 h), at 20°C for a total of 65,000–70,000 VH, using linear 24 cm strips (pH 4–7) in an IPGphore system (GE Healthcare). Vertical SDS-PAGE (12%) was run at a constant wattage (2 W per strip for 45 m, 9 W per strip for 10 h) and was stained with Coomassie blue G-250 (Thermo Scientific) according to the manufacturer’s instructions.
9
Acrosome Reaction Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AR status was evaluated both in NC and CAP sperm samples. Induced AR was obtained by incubating the cells with 10 μM of the calcium ionophore A23187 for 30 min prior the end of the capacitation process. Mitochondrial inhibitors were added to CAP media with and without A23187, and 2.5 μM Antimycin A (AA) (Sigma Aldrich, St. Louis, MO) or 2.5 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma-Aldrich). For all conditions 15 μl samples were placed onto glass slides, fixed in 4% paraformaldehyde for 30 min and washed twice with phosphate buffered saline (PBS). After washing, slides were incubated with 0.22% Coomassie stain (Coomassie Blue G-250; Thermo Scientific, Massachusetts), 50% methanol, 10% glacial acetic acid, 40% water for 2 min. Excess dye was removed by washing thoroughly using distilled water. Slides were air-dried and coverslips were placed on slides using mounting medium at room temperature. Stained sperm were examined under bright field microscopy at 400X (Nikon E100, Japan) to verify the percentage of sperm that had undergone AR. A minimum of 200 sperm was evaluated in each experiment.
10
Hb Cleavage Map by R. prolixus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The AM and PM contents from unfed, 6 h, and 1, 2, 7, and 14 days postfed insects, were treated with EDTA-free protease inhibitor cocktail. Subsequently, the samples were denatured by adding Laemmli 4X buffer and heated at 99 °C for 5 min in order to be analyzed by SDS-PAGE electrophoresis on a 10 to 20% Tris-Tricine gradient gel under reducing conditions. Gel was stained with Coomassie Blue G-250 (Thermo Fisher Scientific Inc), or silver (Serva). Concurrently, the digestive content of AM, specifically at 14 days postfeeding, underwent filtration using a 10 kDa MWCO ultrafiltration column. The run-through, containing small molecules, was then subjected to MS/MS analysis as described previously (Mascot was set up to search the R. prolixus_230123 (18,471 entries) and Oryctolagus cuniculus_230418 (59,928 entries) databases, with nonspecific enzyme digestion parameter). The identified peptides will allow to reconstitute the in vivo cleavage map of Hb by R. prolixus peptidases.
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!