Complete HL-5 and defined SIH (-arginine; -lysine) growth media and unlabelled L -arginine and L -lysine were from Formedium. Labelled K8 L -lysine and R6 L -arginine were from Cambridge Isotope Laboratories. Complete EDTA-free protease inhibitor cocktail was from Roche. Turbo DNase was from Ambion. Novex NuPAGE 4–12% gradient bis-tris gels with MOPS SDS running buffer and Novex colloidal blue staining kit were from Invitrogen. Trypsin Gold was from Promega. LavaPep peptide quantification kit was from Gel Company. ZipTip C-18 peptide purification microcolumns and high fidelity KOD hot start DNA polymerase were from Merck Millipore. Restriction enzymes, T4 DNA ligase and Antarctic phosphatase were from New England Biolabs. Plasmid miniprep kit and PCR clean-up kit were from Qiagen. Krypton fluorescent protein stain and Nunc Lab-Tek chambered cover glass plates for cell imaging were from Thermo Scientific. All the other reagents were obtained from Sigma-Aldrich.
Krypton fluorescent protein stain
Manufactured by Thermo Fisher Scientific
Krypton fluorescent protein stain is a fluorescent dye used to label and detect proteins in gel electrophoresis and Western blot applications. It binds to proteins and emits fluorescence upon excitation, allowing for visualization and quantification of protein bands.
Automatically generated - may contain errors
Lab products found in correlation
Novex colloidal blue staining kit, by Thermo Fisher Scientific (2 mentions)
Plasmid miniprep kit, by Qiagen (2 mentions)
Complete hl 5 and defined sih arginine lysine growth media, by Formedium (2 mentions)
Unlabelled l arginine, by Formedium (2 mentions)
L lysine, by Formedium (2 mentions)
Labelled k8 l lysine, by Cambridge Isotopes (2 mentions)
R6 l arginine, by Cambridge Isotopes (2 mentions)
Novex nupage 4 12 gradient bis tris gels with mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Ziptip c 18 peptide purification microcolumns, by Merck Group (2 mentions)
High fidelity kod hot start dna polymerase, by Merck Group (2 mentions)
Nunc lab tek chambered cover glass plates for cell imaging, by Thermo Fisher Scientific (2 mentions)
Pcr cleanup kit, by Qiagen (2 mentions)
Antarctic phosphatase, by New England Biolabs (2 mentions)
Trypsin gold, by Promega (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Amersham typhoon biomolecular imager, by GE Healthcare (1 mentions)
Mark12, by Thermo Fisher Scientific (1 mentions)
Typhoon 9400, by Cytiva (1 mentions)
Complete edta free protease inhibitors cocktail, by Roche (1 mentions)
4 protocols using krypton fluorescent protein stain
1
Proteome Analysis of SIH Cell Lines
2
Protein Extraction and Quantification
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SF was cleared for cellular debris by centrifugation at 600×g for 15 min at 20 °C, before storage at -80 °C. After thawing, the samples were centrifuged at 20,000×g for 10 min at 4 °C. The protein concentration was measured with the Bicinchonic Acid (BCA) Protein Assay (Thermo Fisher Scientific, Waltham, MA) according the manufactures instruction. Five µg protein in SDS-samples buffer (Expedeon, San Diego, CA) were separated by 12% SDS-polyacrylamide gel electrophoresis (Expedeon). As molecular weight standard, 2.5 µl Mark12® (Thermo Fisher Scientific) was used. The proteins were stained with Krypton™ Fluorescent Protein Stain (Thermo Fisher Scientific) according the manufactures instruction and scanned on an Amersham Typhoon Biomolecular Imager (GE Healthcare, Chicago, IL).
3
Photoinactivation of HγD-Crystallin
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Samples
of HγD-Crys
were prepared at 1 mg/mL in 1× reaction buffer (100 mM Na2PO4 and 1 mM EDTA, pH 7). Samples were irradiated
at room temperature in a quartz cuvette (Starna Group) using a UVP
Inc. UVLMS-38 lamp equipped with a 302 nm midrange bulb delivering
a range of UVA/UVB light. UVR dose delivery was set to 2 mW/cm2 by adjusting the cuvette’s distance to the lamp and
was determined before each experiment by a UVX radiometer with midrange
UVX-31 sensor. Turbidity readings at OD600 on a Cary UV/vis
spectrometer were taken at regular time points during irradiation.
Samples removed and analyzed via SDS-PAGE were reduced and boiled
and electrophoresed through 14% acrylamide gels at 170 V for 1 h;
gels were stained using Krypton fluorescent protein stain (Thermo
Fisher Scientific) and imaged on a Typhoon 9400 (Amersham Biosciences).
Gel band quantification was performed using ImageJ.
of HγD-Crys
were prepared at 1 mg/mL in 1× reaction buffer (100 mM Na2PO4 and 1 mM EDTA, pH 7). Samples were irradiated
at room temperature in a quartz cuvette (Starna Group) using a UVP
Inc. UVLMS-38 lamp equipped with a 302 nm midrange bulb delivering
a range of UVA/UVB light. UVR dose delivery was set to 2 mW/cm2 by adjusting the cuvette’s distance to the lamp and
was determined before each experiment by a UVX radiometer with midrange
UVX-31 sensor. Turbidity readings at OD600 on a Cary UV/vis
spectrometer were taken at regular time points during irradiation.
Samples removed and analyzed via SDS-PAGE were reduced and boiled
and electrophoresed through 14% acrylamide gels at 170 V for 1 h;
gels were stained using Krypton fluorescent protein stain (Thermo
Fisher Scientific) and imaged on a Typhoon 9400 (Amersham Biosciences).
Gel band quantification was performed using ImageJ.
4
Quantitative Mass Spectrometry Proteomics
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