To visualise all proteins, gels were fixed in 40% ethanol (v/v)/10% acetic acid (v/v) in Milli-Q water and stained with Flamingo Fluorescent Stain (Bio-Rad Laboratories) according to the manufacturer's instructions. Gels were destained in 0.1% (v/v) Tween 20 in Milli-Q water for 10 minutes prior to imaging. Gels were scanned using a Typhoon Trio Variable Mode Imager using a green laser (532 nm) excitation source and 610 ± 30 nm bandpass emission filter.
Flamingo fluorescent stain
Manufactured by Bio-Rad
Sourced in United States
Flamingo fluorescent stain is a laboratory reagent used to detect and visualize proteins in electrophoresis gels. It is a sensitive fluorescent dye that binds to proteins, allowing their detection and analysis under ultraviolet (UV) or blue light illumination.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Bio-Rad (1 mentions)
7 cm ipg strips, by Bio-Rad (1 mentions)
Laemelli sample buffer, by Bio-Rad (1 mentions)
Bio spin 6 chromatographycolumns, by Bio-Rad (1 mentions)
Criterion tgx precast gel, by Bio-Rad (1 mentions)
Trypsin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Molecular imager pharosfx plus system, by Bio-Rad (1 mentions)
Criterion tgx, by Bio-Rad (1 mentions)
6 protocols using flamingo fluorescent stain
1
Fluorescent Protein Visualization Protocol
2
Stoichiometric Analysis of AAV5 Subunits
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Stoichiometric analysis of AAV5 VP subunits was performed via SDS-PAGE of the affinity-purified sample. The purified vector was serially diluted within 1 log range of the VGs, with the resulting amount loaded per well in the range of 2.2 × 108–2.2 × 109 VGs. After the run, the gel was stained with Flamingo fluorescent stain, per the manufacturer’s protocol (Bio-Rad Laboratories, Hercules, California). This stain was selected because it offers similar sensitivity and better linear range of quantification in comparison to silver stain. The stained gel was visualized via the ChemiDoc Imager, and the densitometric analysis of the resolved bands was performed using the Image Lab software suite. The linear range of quantification was established by plotting the VP band intensity as a function of VG amount. The best possible linearity between VP band intensity and VG was found to be in the concentration range 5.5 × 108–7.3 × 108 VG loaded (Figure S5 ). Also within this range, the VP3 band intensity was normalized—arbitrarily set to 10—and the relative band intensities of the three VP subunits were determined.
3
SDS-PAGE Protein Analysis
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Purified protein pattern was analyzed through a 7% SDS/PAGE. The protein was stained with Flamingo™ fluorescent stain (Bio Rad Mexico, CDMX, Mexico). Relative molecular mass (Mr) was calculated by comparison with the migration of the standard proteins (Bio‐Rad, SDS/PAGE Broad Range, 161–0317).
4
Isoelectric Focusing and SDS-PAGE for Protein Analysis
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Samples containing 100 µg protein were passively rehydrated onto 7 cm IPG strips (Bio-Rad, Hercules, CA, USA) with a pH range 4–7 for 12–16 h, followed by isoelectric focusing through the three-step protocol: the initial voltage was limited to 250 V for 10 min then increased to 3,500 V for 2,800 Vhr, then continued at 3,500 V to 3,700 Vhr. The current was limited to 50 µA per strip at 20 °C. After isoelectric focusing, the strips were first equilibrated in equilibration (EQ) buffer containing 2% 1,4-dithiothreitol (Bio-Rad, Hercules, CA, USA) for 15 min, followed by another 15 min equilibration in the EQ buffer containing 2.5% Iodoacetamide (Bio-Rad, Hercules, CA, USA) with shaking. The proteins were resolved on a 10% SDS-PAGE for the albumin-containing samples and 12% SDS-PAGE for the albumin-depleted samples. The gels were fixed in methanol/acetic acid/water solution (4:1:5) for 2 h, followed by 3 h of staining with 1X Flamingo fluorescent stain (Bio-Rad, Hercules, CA, USA).
5
Quantification of Protein Concentrations
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The protein concentrations of E1, E2, E3 and E4 samples were ascertained using SDS-PAGE. Samples diluted to 1:10 and 1:50 and a serial dilution of albumin (1 mg/mL) were mixed with Laemelli sample Buffer (Bio-Rad). Forty microliters of each sample was loaded onto 26-well Criterion™ TGX™ Precast Gels (Bio-Rad), and run at 160 V for 3 min. The gel was fixed and then stained with Flamingo™ Fluorescent stain (Bio-Rad). The density of bands was quantified using Quantity One software (v4.6.1, Bio-Rad) based on the BSA standard curve.
The E4 sample contained salt which was subsequently removed using Bio-spin 6 chromatography columns (Bio-Rad), and then dilutions using ratios of 1:2, 1:4 and 1:8 were carried out on the E4 sample and subjected to SDS = PAGE which showed the salt had been removed.
Approximately 100 µg of protein was diluted with 50 mM NH4HCO3 to adjust the urea concentration to 1 M. Trypsin (1 µL, MS grade, 1 µg/µL, Sigma-Aldrich, St. Louis, MO, USA) was added to each tube, and the sample tubes were incubated overnight at 37 °C. The digested samples were centrifuged at maximum speed for 5 min prior to solid phase extraction using HLB columns (Silicycle®, Quebec, QC, Canada) to recover the peptides. Samples were concentrated to approximately 15 µL (E1), or 25 µL (E2, E3 and E4).
The E4 sample contained salt which was subsequently removed using Bio-spin 6 chromatography columns (Bio-Rad), and then dilutions using ratios of 1:2, 1:4 and 1:8 were carried out on the E4 sample and subjected to SDS = PAGE which showed the salt had been removed.
Approximately 100 µg of protein was diluted with 50 mM NH4HCO3 to adjust the urea concentration to 1 M. Trypsin (1 µL, MS grade, 1 µg/µL, Sigma-Aldrich, St. Louis, MO, USA) was added to each tube, and the sample tubes were incubated overnight at 37 °C. The digested samples were centrifuged at maximum speed for 5 min prior to solid phase extraction using HLB columns (Silicycle®, Quebec, QC, Canada) to recover the peptides. Samples were concentrated to approximately 15 µL (E1), or 25 µL (E2, E3 and E4).
6
2D-PAGE Venom Proteome Analysis
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300 µg samples of venom, collected by electrical stimulation or manual dissection, were resuspended in 100 µl 7 M urea, 2 M thiourea, 1% (v/v) C7BzO, 50 mM Tris HCl pH 8.8 before reduction and alkylation of disulphide bonds with 5 mM tributylphosphine (TBP) and 20 mM acrylamide monomers for 90 mins. The sample was subjected to 2D-PAGE as previously described 40 . Briefly, sample was fractionated by isoelectric focusing on an 11 cm pH 3-10 IPG strip (Bio-Rad, Hercules CA) for 100 kVh and then further separated by molecular size by SDS-PAGE on a 4-20% Tris-glycine gel (Criterion TGX, Bio-Rad, NSW, Australia). The gel was then placed in a fixing solution containing 40% (v/v) methanol and 10% (v/v) acetic acid for 30 mins at room temperature before staining with Flamingo fluorescent stain (Bio-Rad) for 1 h. After staining, the gel was scanned using a Molecular Imager PharosFX Plus system (Bio-Rad) with QuantityOne® software. In order to perform nanoLC-ESI-QTOF MS/MS, gels were additionally stained with Coomassie Blue G250 overnight then destained with 1% (v/v) acetic acid for 2 h.
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