Human muscle tissue was extracted in: 50 mM Tris pH 6.8, 1% EDTA, 10% SDS, 5% beta-mercaptoethanol, 10% glycerol with protease inhibitors. After the addition of bromophenol blue and after boiling, the extract was loaded, either as a single strip or in formed wells, and subjected to SDS-PAGE using 4 to 12% polyacrylamide gradient gels and transferred to nitrocellulose membranes (Protan BA85, Whatman). After blocking non-specific sites with 5% skimmed milk protein, membranes were incubated for 1 hour with monoclonal antibody (1/50), followed by washing and incubation for 1 hour with peroxidase-labelled rabbit anti-mouse immunoglobulins (1/1000, Dakopatts, Denmark). Antibody reacting bands were visualized with West Pico or West Femto chemiluminescent detection systems (Pierce, Thermofisher). Prestained Mr markers were EZ-Run 10–170 kD or PageRuler Plus 10–250 kD (both Thermofisher).
Ez run
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The EZ-Run is a laboratory equipment product designed to perform electrophoresis. It is used to separate and analyze biomolecules, such as proteins or nucleic acids, based on their size and charge.
Automatically generated - may contain errors
Lab products found in correlation
Mops running buffer, by Thermo Fisher Scientific (4 mentions)
Pageruler plus, by Thermo Fisher Scientific (1 mentions)
Protan ba85, by Cytiva (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Invitrolon pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal western blot enhancer, by Thermo Fisher Scientific (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Bolt gel, by Thermo Fisher Scientific (1 mentions)
Anti bcl xl, by New England Biolabs (1 mentions)
Tricine gel, by Thermo Fisher Scientific (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Seeblue plus2, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using ez run
1
Western Blot for Muscle Proteins
2
Protein Detection and Western Blot Analysis
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TCF7 was detected with the mixture of antibodies 1D2 and 2E9 (Novus Biologicals, Littleton, CO). The MSI1 protein was detected by monoclonal antibody (EP1302) (Abcam, Cambridge, MA). Human actin was stained by monoclonal antibody AC-15 (ab6276) (Abcam). The cells were lysed in buffers supplemented with a set of inhibitors which included: 2× concentrated complete protease inhibitor cocktail (Roche Diagnostics, Indianopolis, IN), 2 mM phenylmethylsulfonyl fluoride, and aprotinin (Sigma-Aldrich, St. Louis, MO). Prestained molecular weight markers EZ-RUN from Thermo Fisher Scientific (Walthman, MA) were employed to determine molecular weights. Proteins were separated on NuPAGE 4–12% Bis-Tris gels, electroblotted to Invitrolon PVDF membrane (Invitrogen, Carlsbad, CA) and visualized with SuperSignal West Dura Chemiluminescent Substrate with (MSI1) or without (TCF7) SuperSignal Western blot enhancer (Thermo Fisher Scientific).
3
Western Blot Analysis of Apoptosis Regulators
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Cells were harvested with accutase treatment when adequate, washed with PBS, lysed and heated at 95 °C for 5 min (Laemmli buffer), 70 °C for 10 min (Bolt sample buffer) or 85 °C for 2 min (Tricine sample buffer). Extracts were separated by SDS-PAGE either on 12.5% gels (EZ-run, Fisher Scientific, Schwerte, Germany), 4–12% Bolt gels (Life Technologies) or 16% Tricine gels (Life Technologies), and proteins were transferred onto nitrocellulose membranes (0.2 μm). Membranes were probed with anti-mouse A1 (rat monoclonal, kindly provided by Marco Herold, WEHI, Melbourne, Australia), anti-human A1 (rabbit polyclonal, kindly provided by Jannie Borst), anti-mouse Mcl-1 (Rockland, Limerick, PA, USA, #600-401-394), anti-human Mcl-1 (BD, Biosciences, Heidelberg, Germany, #559027), anti-mouse Bcl-2 (BD, clone 3F11), anti-Bcl-xL (NEB, Frankfurt, Germany, clone 54H6) or anti-GAPDH (Millipore, Darmstadt, Germany, clone 6C5) or anti-β-actin (Sigma, clone AC-15) antibodies. Proteins were visualized using peroxidase-conjugated anti-rabbit (Sigma), anti-mouse (Dianova), anti-hamster (Dianova) or anti-rat (NEB) antibodies by enhanced chemoluminescence detection (ECL Prime, GE Healthcare, Dornstadt, Germany; SuperSignal West Femto Substrate, Pierce, Fisher Scientific, Schwerte, Germany).
4
PEGylation of Yeast Enolase
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Aliquots (100 μL) of denatured
yeast enolase (1 mg/mL, 11 μM) were treated with varying concentrations
of TCEP or THPP (1–10 mM) and incubated for 45 min at 25 °C.
2 kDa-PEG maleimide (1 mM) was subsequently added to the enolase solutions
at each phosphine concentration and incubated at 37 °C for 18
h. Samples (15 μL) were taken from each of the reactions and
added to the Laemmli sample buffer (15 μL) and heated (85 °C,
8 min). Aliquots (9 μL) of these solutions were loaded into
a precast gradient gel (4–12% Bis-Tris, Invitrogen) along with
a protein ladder (EZ-Run, Fisher Scientific) and resolved by SDS Page
electrophoresis [MOPS running buffer (Invitrogen), 180 V, 60 min].
The precast gels were stained by the Coomassie solution and destained
using a water/ethanol/acetic acid (16:3:1) solution. The gel was then
scanned using a LI-COR Odyssey CLx to quantify PEGylated enolase (Image
Studio Lite).
yeast enolase (1 mg/mL, 11 μM) were treated with varying concentrations
of TCEP or THPP (1–10 mM) and incubated for 45 min at 25 °C.
2 kDa-PEG maleimide (1 mM) was subsequently added to the enolase solutions
at each phosphine concentration and incubated at 37 °C for 18
h. Samples (15 μL) were taken from each of the reactions and
added to the Laemmli sample buffer (15 μL) and heated (85 °C,
8 min). Aliquots (9 μL) of these solutions were loaded into
a precast gradient gel (4–12% Bis-Tris, Invitrogen) along with
a protein ladder (EZ-Run, Fisher Scientific) and resolved by SDS Page
electrophoresis [MOPS running buffer (Invitrogen), 180 V, 60 min].
The precast gels were stained by the Coomassie solution and destained
using a water/ethanol/acetic acid (16:3:1) solution. The gel was then
scanned using a LI-COR Odyssey CLx to quantify PEGylated enolase (Image
Studio Lite).
5
Quantitative Analysis of PEGylated Yeast Enolase
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Aliquots (100 μL) of denatured yeast enolase (1 mg/mL, 11 μM)
were treated with varying concentrations of TCEP or THPP (1–10
mM) and incubated for 45 min at 25 °C. Samples were then treated
with PEG-azide7 (100 mM) and held for 1 hour at 37 °C.
2 kDa-PEG maleimide (1 mM) was subsequently added to these solutions
and incubated at 37 °C for 18 h. Samples (15 μL) were taken
from each of the reactions and added to the Laemmli sample buffer
(15 μL) and heated (85 °C, 8 min). Aliquots (9 μL)
of these solutions were loaded into a precast gradient gel (4–12%
Bis-Tris, Invitrogen) along with a protein ladder (EZ-Run, Fisher
Scientific) and resolved by SDS Page electrophoresis [MOPS running
buffer (Invitrogen), 180 V, 60 min]. The precast gels were stained
by the Coomassie solution and destained using a water/ethanol/acetic
acid (16:3:1) solution. The gel was then scanned using a LI-COR Odyssey
CLx to quantify PEGylated enolase (Image Studio Lite).
were treated with varying concentrations of TCEP or THPP (1–10
mM) and incubated for 45 min at 25 °C. Samples were then treated
with PEG-azide
2 kDa-PEG maleimide (1 mM) was subsequently added to these solutions
and incubated at 37 °C for 18 h. Samples (15 μL) were taken
from each of the reactions and added to the Laemmli sample buffer
(15 μL) and heated (85 °C, 8 min). Aliquots (9 μL)
of these solutions were loaded into a precast gradient gel (4–12%
Bis-Tris, Invitrogen) along with a protein ladder (EZ-Run, Fisher
Scientific) and resolved by SDS Page electrophoresis [MOPS running
buffer (Invitrogen), 180 V, 60 min]. The precast gels were stained
by the Coomassie solution and destained using a water/ethanol/acetic
acid (16:3:1) solution. The gel was then scanned using a LI-COR Odyssey
CLx to quantify PEGylated enolase (Image Studio Lite).
6
Quantitative Enolase Labeling Assay
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Aliquots
(100 μL) of denatured yeast enolase (1 mg/mL, 11 μM) were
treated with varying concentrations of TCEP or THPP (1–10 mM)
and incubated for 45 min at 25 °C. Maleimide fluorescein11 (1 mM) was subsequently added to the enolase solutions
at each phosphine concentration and incubated at 37 °C for 18
h. Samples (15 μL) were taken from each of the reactions and
added to the Laemmli sample buffer (15 μL) and heated (85 °C,
8 min). Aliquots (9 μL) of these solutions were then loaded
into a precast gradient gel (4–12% Bis-Tris, Invitrogen) along
with a protein ladder (EZ-Run, Fisher Scientific) and resolved by
SDS Page electrophoresis [MOPS running buffer (Invitrogen), 180 V,
60 min]. The precast gels were first stained by Coomassie solution
and destained using a water/ethanol/acetic acid (16:3:1) solution
to confirm equal protein loading. Fluorescence was then visualized
at 525 nm (Dark Reader).
(100 μL) of denatured yeast enolase (1 mg/mL, 11 μM) were
treated with varying concentrations of TCEP or THPP (1–10 mM)
and incubated for 45 min at 25 °C. Maleimide fluorescein
at each phosphine concentration and incubated at 37 °C for 18
h. Samples (15 μL) were taken from each of the reactions and
added to the Laemmli sample buffer (15 μL) and heated (85 °C,
8 min). Aliquots (9 μL) of these solutions were then loaded
into a precast gradient gel (4–12% Bis-Tris, Invitrogen) along
with a protein ladder (EZ-Run, Fisher Scientific) and resolved by
SDS Page electrophoresis [MOPS running buffer (Invitrogen), 180 V,
60 min]. The precast gels were first stained by Coomassie solution
and destained using a water/ethanol/acetic acid (16:3:1) solution
to confirm equal protein loading. Fluorescence was then visualized
at 525 nm (Dark Reader).
7
Western Blot Protein Analysis
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Protein samples were mixed with NuPAGE-LDS sample buffer (Invitrogen) containing 50 mM dithiothreitol (DTT), then denatured by heating to 70°C for 10 min and resolved by electrophoresis on precast 10% Tris–glycine polyacrylamide gels (Novex). Molecular weight markers (SeeBlue Plus2 [Invitrogen] and EZ-Run [Fisher Scientific]) were resolved in parallel to determine protein sizes. Proteins were transferred from gels onto nitrocellulose membranes by using an iBlot dry transfer apparatus (Thermo, Fisher). Membranes were blocked by incubation in 5% Carnation dry milk dissolved in PBS–0.2% Tween 20 (PBST) prior to incubation with primary antibody diluted in milk-PBST solution. Membranes were washed with PBST before incubating with HRP-conjugated secondary antibody diluted in milk-PBST solution. Membranes were washed with PBST and developed using the SuperSignal West Pico chemiluminescent substrate (Pierce). Signal was detected by exposing membranes to BioExcell X-ray film or by using the Azure Series c500 infrared imaging system. Bands of interest were normalized to the loading control, PCNA.
8
Yeast Enolase Redox Labeling Protocol
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Aliquots (100 μL) of
denatured yeast enolase
(1 mg/mL, 11 μM) were treated with varying concentrations of
TCEP or THPP (1–10 mM) and incubated for 45 min at 25 °C.
Samples were then treated with PEG-azide7 (100 mM) and
held for 1 hour at 37 °C. Maleimide-fluorescein (1 mM) was subsequently
added to these solutions and incubated at 37 °C for 18 h. Samples
(15 μL) were taken from each of the reactions and added to the
Laemmli sample buffer (15 μL) and heated (85 °C, 8 min).
Aliquots (9 μL) of these solutions were loaded into a precast
gradient gel (4–12% Bis-Tris, Invitrogen) along with a protein
ladder (EZ-Run, Fisher Scientific) and resolved by SDS Page electrophoresis
[MOPS running buffer (Invitrogen), 180 V, 60 min]. The precast gels
were stained by the Coomassie solution and destained using a water/ethanol/acetic
acid (16:3:1) solution to confirm equal protein loading. Fluorescence
was then visualized at 525 nm (Dark Reader).
denatured yeast enolase
(1 mg/mL, 11 μM) were treated with varying concentrations of
TCEP or THPP (1–10 mM) and incubated for 45 min at 25 °C.
Samples were then treated with PEG-azide
held for 1 hour at 37 °C. Maleimide-fluorescein (1 mM) was subsequently
added to these solutions and incubated at 37 °C for 18 h. Samples
(15 μL) were taken from each of the reactions and added to the
Laemmli sample buffer (15 μL) and heated (85 °C, 8 min).
Aliquots (9 μL) of these solutions were loaded into a precast
gradient gel (4–12% Bis-Tris, Invitrogen) along with a protein
ladder (EZ-Run, Fisher Scientific) and resolved by SDS Page electrophoresis
[MOPS running buffer (Invitrogen), 180 V, 60 min]. The precast gels
were stained by the Coomassie solution and destained using a water/ethanol/acetic
acid (16:3:1) solution to confirm equal protein loading. Fluorescence
was then visualized at 525 nm (Dark Reader).
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