To evaluate if purified lipopeptides prevent hemolysis by impairing α-hemolysin oligomerization, an SDS-PAGE was performed as described below. The active lipopeptide was mixed with 2.5 µg of alpha-hemolysin (Sigma-Aldrich, St. Louis, USA) at 0.5, 0.25, and 0.125 mg/mL. To promote oligomerization, 5 mM of deoxycholate was added to the samples, and the mixtures were incubated at 22 °C for 20 min27 (link). TruPAGE LDS sample buffer 4× (Merck, Darmstadt, Germany) was then added to the samples at 1× final concentration and the mixtures were incubated at 50 °C for 10 min27 (link). Twenty-five µL of each reaction mixture was loaded onto NuPAGE 12% Bis–Tris Gel (Invitrogen, Massachusetts, EUA). Ten µL of the SeeBlue Plus2 Pre-stained (Invitrogen, Massachusetts, EUA) were applied as the protein standard. The gel was run at 100 V for 50 min using MES buffer (Invitrogen, Massachusetts, EUA). Gels were stained using the EZblue Gel Staining Reagent (Sigma, Missouri, EUA) overnight.
Seeblue plus2 pre stained
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SeeBlue Plus2 Pre-Stained is a protein molecular weight marker used for protein electrophoresis. It provides a visual reference for tracking the migration of proteins during SDS-PAGE.
Automatically generated - may contain errors
Lab products found in correlation
Simplyblue safestain, by Thermo Fisher Scientific (2 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Ezblue gel staining reagent, by Merck Group (1 mentions)
Alpha hemolysin, by Merck Group (1 mentions)
Nupage 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nupage 12 bis tris, by Thermo Fisher Scientific (1 mentions)
Sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Magicmark xp, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Nupage system, by Thermo Fisher Scientific (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nupage reducing agent, by Thermo Fisher Scientific (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using seeblue plus2 pre stained
1
Lipopeptide Inhibition of α-Hemolysin Oligomerization
2
Zinc-Induced Fluorescence of Bilin Chromophores
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Samples equivalent to OD620 = 0.5 were analysed on NuPAGE 12% Bis-Tris (Invitrogen) protein gel set to run at 150 V for 1 h. SeeBlue Plus2 Pre-Stained (Invitrogen) protein standard was used as a ladder. The gels were incubated in 100 mM zinc sulfate solution for 10 min and visualized under UV light for the presence of zinc-induced fluorescence of bilin chromophores (Raps, 1990 (link)). The gels were further stained with 1% (v/v) Coomassie Blue in acetic acid/methanol. For the bands extracted and purified from sucrose gradients (section 2.9 ) Coomassie staining was not able to clearly visualize the bands. Therefore, the gel was re-stained using silver staining as described in Heukeshoven and Dernick (1985) .
3
Venom Protein Separation Protocol
4
Transfection Optimization for Cell Lines
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TO cells were split 2:3 the day before transfection. The cells were transfected at 80% confluence using the 10 μl Neon transfection system (Life technologies) with buffer R and at a pulse voltage of 1200, pulse width 20 ms, 2 pulses. 450,000 cells were used for each transfection. For each well a total of 350 ng plasmid were used. After transfection the cells were seeded in 48-well plates in triplicates with MEM growth medium containing 12% FBS without antibiotics, and incubated with CO2 at 20 °C. Cells were harvested 48 h post transfection in 40 μl 2× SDS buffer. 15–20 μl of the samples were loaded in each well of a precast 4–12% gradient NuPAGE Novex Bis-Tris gel and subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) with 1× MOPS (Invitrogen) for 45 min at 200 V and 120 mA. The markers MagicMark™ XP (Invitrogen) and SeeBlue Plus2 Prestained (Invitrogen) were simultaneously loaded for molecular weight estimations. Western blotting of the separated proteins to a polyvinylidene difluoride (PVDF) membrane (Millipore) was performed using the Invitrogen NuPAGE system according to the manufacturer's instructions. An anti-Flag antibody (1:3000 dilution) (Sigma) for detection of Flag-tagged proteins were used and goat anti-mouse-HRP antibody (1:5000) (Santa Cruz Biotechnology) as a secondary antibody.
5
Protein Quantification and SDS-PAGE Analysis
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Proteins were quantified by the Pierce 660 nm Protein Assay (Thermo Fisher Scientific) according to the manufacturer’s manual. The adsorption was measured with a Tecan Infinite M1000 plate reader using bovine serum albumin (Merck, Kenilworth, NJ, USA) for a calibration curve.
After quantification, the proteins were separated with SDS-PAGE and stained by Silver Staining according to the manufacturer’s recommendations. A total of 2 µg protein in 26 µL total volume was mixed with 4 µL NuPage Reducing Agent and 10 mL of NuPage LDS Sample Buffer (Thermo Fisher Scientific). The sample was loaded on a 10% Bis-Tris-Protein Gel using SeeBlue Plus2 Pre-Stained (Invitrogen, Waltham, MA, USA) as a molecular marker and NuPage MES SDS Running Buffer. Gels were stained with Pierce Silver Stain Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.
After quantification, the proteins were separated with SDS-PAGE and stained by Silver Staining according to the manufacturer’s recommendations. A total of 2 µg protein in 26 µL total volume was mixed with 4 µL NuPage Reducing Agent and 10 mL of NuPage LDS Sample Buffer (Thermo Fisher Scientific). The sample was loaded on a 10% Bis-Tris-Protein Gel using SeeBlue Plus2 Pre-Stained (Invitrogen, Waltham, MA, USA) as a molecular marker and NuPage MES SDS Running Buffer. Gels were stained with Pierce Silver Stain Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.
6
Separation of Hard Corona Proteins
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Hard corona proteins were separated using 10% Bis-Tris-Protein Gels and NuPAGE MES and SDS Running Buffer. Proteins (8 µg in 26 µL) were mixed with NuPage Reducing Agent (4 µL) and NuPage LDS Samples Buffer (10 µL). The gel was run for 1.5 h at 100 V with SeeBlue Plus2 Pre-Stained (Invitrogen) as molecular marker. Gels were stained with SimplyBlue SafeStain overnight and destained with water (all reagents Thermo Fisher Scientific).
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