BHMMs were resuspended in KPi buffer (50 mM KPi, pH 7.8 at 30°C) and deactivated (30 min at 37°C) or activated (kept on ice for 25 min followed by incubation with 1 mM NADH on ice. Membranes were pelleted (17,000 × g, 3 min, 4°C) and exposed thiols were blocked with 0.5 mM NEM (in KPi buffer) for 30 min on ice. Membranes were washed twice with 35 mM cysteine and subsequently once with plain KPi buffer. Then samples were kept either on ice or deactivated at 37°C for 30 min, pelleted (17,000 × g, 3 min, 4°C) and resuspended in 50 μl KPi containing 0.5 mM N-fluorescein maleimide or Cy5-NEM dye (sufficient dye to label 50 μg of unlabeled protein, according to manufacturer) and labelled for 30 min on ice in the dark. Membranes were washed twice with 35 mM cysteine and subsequently once with plain KPi buffer. Next, proteins were extracted with 1% DDM and separated by BN-PAGE. The band corresponding to complex I was excised, cut into very small pieces, sample buffer (4× Laemmli) was added and all liquid and gel-remnants were loaded into the wells of a SDS-PAGE gel and proteins were resolved. The fluorescent labelling in the gel was scanned using an Amersham Typhoon RGB scanner. Then, the gel was fixed (50% methanol and 10% acetic acid) and stained with QC colloidal brilliant blue staining solution (Bio-Rad, UK).
Typhoon rgb scanner
Manufactured by Cytiva
Sourced in United Kingdom
The Typhoon RGB scanner is a versatile tool for high-resolution imaging of various samples, including gels, blots, and microarrays. It is capable of detecting a wide range of fluorescent and chemiluminescent signals using three different excitation wavelengths (green, red, and blue).
Automatically generated - may contain errors
Lab products found in correlation
7 protocols using typhoon rgb scanner
1
Fluorescent Labeling of Complex I
2
Characterization of Complex I Thiols
3
Redox-Sensitive Complex I Proteomic Mapping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BHMMs were activated in KPi buffer (50 mM KPi, pH 7.8 at 30°C; incubated in presence of 0.4 mM NADPH, shaking at RTfor 30 min). Membranes were washed and exposed thiols were blocked with 30 mM NEM (incubating for 30 min at 15°C) in KPi buffer (pH 9). Membranes were washed with 35 mM cysteine and subsequently with plain KPi buffer (pH 7.8). The samples were split into two. One fraction (active) was incubated on ice for 90 min while the other fraction (deactive) was incubated for 90 min at 37°C. Then, membranes were labelled with 0.5 mM N-fluorescein maleimide for 20 min at 15°C in the dark. Membranes were washed with 35 mM cysteine and subsequently twice with plain KPi buffer. Next, proteins were extracted with 1% DDM and separated by BN-PAGE. The band corresponding to complex I was excised, cut into very small pieces, sample buffer (4× Laemmli) was added and all liquid and gel-remnants were loaded into the wells of a SDS-PAGE gel and proteins were resolved. The fluorescent labelling in the gel was scanned using an Amersham Typhoon RGB scanner. Then, the gel was fixed (50% methanol and 10% acetic acid) and stained with QC colloidal brilliant blue staining solution (Bio-Rad, UK) (Galkin et al., 2008 (link)).
4
Primer extension on modified templates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The specificity of dATP insertion opposite oxo-ϵA and subsequent primer elongation were studied on native (ODN11 ; see Supplementary Table S1 for oligodeoxynucleotide (ODN) structures) and modified (ODN12 ) templates by primer extension experiments using primer (ODN13 ) bearing Cy5 residue at 5′-end and four native dNTPs (Supplementary Table S1 ). The reactions were performed by mixing a preliminary annealed primer-template duplex (5 μM) in 1× corresponding reaction buffer, 750 μM each of dNTPs (with or without dATP), and 2 units of either E. coli DNA Polymerase I, Large fragment (Klenow fragment) or T4 DNA polymerase (SibEnzyme, Russia). Primer extension was carried out at 37°C for 4 h, with the analysis of a reaction mixture every hour. Formamide-containing dye was used to terminate the reaction and products were evaluated by 19% denaturing PAGE. The gels were scanned using a Typhoon RGB scanner (Amersham, UK). Bands with the products of the primer extension reaction were cut out, and extension products were eluted and analyzed by mass-spectrometry.
5
Characterization of Complex I Thiols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BHMMs were resuspended in KPi buffer (50 mM KPi, pH 7.8 at 30°C) and deactivated (20 min at 37°C) or activated (kept on ice for 15 min followed by incubation with 1 mM NADH on ice. Membranes were pelleted (17,000 × g, 3 min, 4°C) and exposed thiols were blocked with 10 mM NEM (in KPi buffer) for 15 min on ice. Membranes were washed with 35 mM cysteine and subsequently with plain KPi buffer. Then, membranes were deactivated (20 min at 37°C) and labelled with 0.5 mM N-fluorescein maleimide for 10 min at RT in the dark. Membranes were washed with 35 mM cysteine and subsequently twice with plain KPi buffer. Next, proteins were extracted with 1% DDM and separated by BN-PAGE. The band corresponding to complex I was excised, cut into very small pieces, sample buffer (4× Laemmli) was added and all liquid and gel-remnants were loaded into the wells of a SDS-PAGE gel and proteins were resolved. The fluorescent labelling in the gel was scanned using an Amersham Typhoon RGB scanner. Then, the gel was fixed (50% methanol and 10% acetic acid) and stained with QC colloidal brilliant blue staining solution (Bio-Rad, UK).
6
Redox-Sensitive Complex I Proteomic Mapping
7
Fluorescent Labeling of Complex I
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!