Lysates were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with anti-vinculin (Sigma, 1:10000), anti-ATP6V1A (Abnova, 1:1000), anti-ATP6V0a3 (Novus, Centennial, CO; 1:1000), anti-FLAG (Sigma, 1:2000), anti-GFP (Cell Signaling, 1:1000), anti-T7 (Novagen, 1:10000), anti-tubulin (Sigma, 1:10000), anti-Rac1 (BD Transduction Laboratories, 1:1000), anti-AKT (Cell Signaling, 1:1000), anti-pAKT (S473) (Cell Signaling, 1:500), anti-ERK2 (EMD Millipore, 1:2000), anti-pERK1/2 (Cell Signaling, 1:1000), anti-SLC4A7 (Santa Cruz, 1:200), or anti-KRas (Santa Cruz, 1:500) primary antibodies followed by Alexa Fluor 680 goat anti-mouse IgG (Life Technologies, 1:10000) or IRDye 800CW goat anti-rabbit IgG (Li-Cor, 1:10000) secondary antibodies. Blots were analyzed using an Odyssey Classic imager (Li-Cor).
Odyssey classic imager
Manufactured by LI COR
Sourced in United States
The Odyssey Classic imager is a fluorescence imaging system designed for gel and Western blot analysis. It employs infrared fluorescence detection to enable quantitative, highly sensitive imaging of a variety of biomolecules, including proteins, nucleic acids, and small molecules. The system is capable of two-color detection, allowing for simultaneous visualization and quantification of multiple targets.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti pakt s473, by Cell Signaling Technology (2 mentions)
Anti rac1, by BD (2 mentions)
Anti kras, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 680 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti atp6v1a, by Abnova (2 mentions)
Anti atp6v0a3, by Novus Biologicals (2 mentions)
Anti erk2, by Merck Group (2 mentions)
Anti slc4a7, by Santa Cruz Biotechnology (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (2 mentions)
Anti p erk1 2, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti gfp, by Cell Signaling Technology (2 mentions)
Image studio lite, by LI COR (2 mentions)
Rabbit anti uchl 1, by Abcam (2 mentions)
Irdye 680lt goat anti mouse igg h l, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (2 mentions)
22 protocols using odyssey classic imager
1
Western Blot Analysis of Cellular Proteins
2
Immunoblotting with Tubulin and AML1
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Primary antibodies included a mouse anti-α-tubulin (1:10,000) antibody (12G10, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and a previously described rabbit anti-AML1 (1:500) antibody generated by Covance[23 (link)]. Licor (Lincoln, NE) IRDye 680RD goat anti-mouse IgG and IRDye 800CW goat anti-rabbit IgG secondary antibodies (1:10,000) were used for visualization on a LI-COR Odyssey Classic imager. Image analysis and densitometry were performed using the LI-COR Application Software Version 3.0.
3
Western Blot Analysis of Protein Lysates
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Cells were collected and then lysed using 8 M urea and 100 mM ammonium bicarbonate on ice. Lysate protein concentrations were measured by micro BCATM protein assay kit (Thermo Scientific, USA). After normalization, equal amounts of proteins diluted in 1x LDS loading buffer containing 0.1 M DTT were boiled for 10 minutes at 95°C prior to electrophoresis. After gel electrophoresis, proteins were then transferred onto a nitrocellulose membrane using iBlot 2 Dry Blotting System (Thermo Scientific, USA), blocked with 5% skim milk in 1x TBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies. Following membrane washing, secondary antibodies IRDye 800 goat-anti-rabbit (Li-COR Biosciences, USA) or IRDye 800 goat-anti-mouse (Li-COR Biosciences, USA) were added for 1 hour at room temperature and washed three times with 1x TBST. The blot was then imaged with the Odyssey Classic Imager (Li-COR Biosciences, USA).
4
Western Blot Analysis Protocol
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Samples were separated by an 8% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Protan BA85, 0.45 μm, GE Healthcare) at 300 mA for 2.5 h. The membranes were blocked in 5% milk (skim milk powder, LP0031, Oxiod) in 1× PBS (P1379, Sigma-Aldrich), incubated with a primary antibody diluted in 5% milk in 0.1% PBS-Tween 20 (PBST) for 1 h, washed three times for 10 min in 0.1% PBST, incubated with the secondary antibody diluted in 5% milk in 0.1% PBST for 30 min, and washed three times again in 0.1% PBST. The signal was detected using direct imaging by the Odyssey Classic imager (LI-COR). Intensity of bands was quantified using the Image Studio software. Unmodified blots corresponding to key experiments presented in Figs. 1 –8 can be found in Supplementary Figs. 10 and 11 .
5
Western Blot Protein Detection Protocol
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After proteins were transferred to a
nitrocellulose membrane at 300 mA for 2.5 h, the membranes were blocked
with 5% milk in PBS and incubated with primary antibody diluted in
5% milk in 0.1% PBS-Tween 20 (PBST) for 1 h at RT. After washing with
0.1% PBST three times for 10 min, proteins were incubated with secondary
antibodies diluted in 0.1% PBST for 30 min and washed three times
again in 0.1% PBST. The signal was detected using direct imaging using
an Odyssey Classic imager (LI-COR).
nitrocellulose membrane at 300 mA for 2.5 h, the membranes were blocked
with 5% milk in PBS and incubated with primary antibody diluted in
5% milk in 0.1% PBS-Tween 20 (PBST) for 1 h at RT. After washing with
0.1% PBST three times for 10 min, proteins were incubated with secondary
antibodies diluted in 0.1% PBST for 30 min and washed three times
again in 0.1% PBST. The signal was detected using direct imaging using
an Odyssey Classic imager (LI-COR).
6
Western Blot Analysis of AVICs and AVs
7
Immunoblot detection of caspase-1 and tissue factor
8
Western Blot Analysis of Cellular Proteins
9
Quantitative Western Blot and DNA Damage Assay
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Cells were seeded into 12-well format (250.000 cells/well), treated
with indicated drugs at 10 μM for 2 h. Subsequently, drugs were
removed by extensive washing and cells were collected and processed
immediately for the assays. For Western blot, cells were lysed directly
in SDS-sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol,
60mM Tris–HCl pH 6.8 and 0.01% bromophenol blue). Samples were
separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to a PVDF membrane (Immobilon-P, 0.45 μm,
Millipore). Blocking of the filters and antibody incubations were
done in PBS supplemented with 0.1 (v/v)% Tween and 5% (w/v) milk powder
(Skim milk powder, LP0031, Oxiod). Blots were imaged by the Odyssey
Classic imager (Li-Cor). Intensity of bands was quantified using ImageJ
or Image Studio software. For CFGE: DNA double strand breaks were
quantified by constant-field gel electrophoresis as described.28 (link) Images were quantified using ImageJ software.
with indicated drugs at 10 μM for 2 h. Subsequently, drugs were
removed by extensive washing and cells were collected and processed
immediately for the assays. For Western blot, cells were lysed directly
in SDS-sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol,
60mM Tris–HCl pH 6.8 and 0.01% bromophenol blue). Samples were
separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to a PVDF membrane (Immobilon-P, 0.45 μm,
Millipore). Blocking of the filters and antibody incubations were
done in PBS supplemented with 0.1 (v/v)% Tween and 5% (w/v) milk powder
(Skim milk powder, LP0031, Oxiod). Blots were imaged by the Odyssey
Classic imager (Li-Cor). Intensity of bands was quantified using ImageJ
or Image Studio software. For CFGE: DNA double strand breaks were
quantified by constant-field gel electrophoresis as described.28 (link) Images were quantified using ImageJ software.
10
Western Blot Analysis of PARK7 and UCHL1
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The following antibodies were used for detection
of endogenous protein by Western blot analysis in a 1:1000 dilution:
rabbit anti-PARK7 (Abcam, Cat# ab18257) and rabbit anti-UCHL1(Abcam,
Cat# ab27053). Mouse anti-β-actin (Sigma-Aldrich, Cat# A5441)
was used as a loading control in a 1:10,000 dilution for the Western
blot analysis. Secondary IRDye 800CW goat anti-rabbit IgG (H + L)
(Li-COR, Cat# 926-32211, 1:5000) and IRDye 680LT goat anti-mouse IgG
(H + L) (Li-COR, Cat# 926-68020, 1:20,000) were used for detection
using an Odyssey Classic imager (LI-COR).
of endogenous protein by Western blot analysis in a 1:1000 dilution:
rabbit anti-PARK7 (Abcam, Cat# ab18257) and rabbit anti-UCHL1(Abcam,
Cat# ab27053). Mouse anti-β-actin (Sigma-Aldrich, Cat# A5441)
was used as a loading control in a 1:10,000 dilution for the Western
blot analysis. Secondary IRDye 800CW goat anti-rabbit IgG (H + L)
(Li-COR, Cat# 926-32211, 1:5000) and IRDye 680LT goat anti-mouse IgG
(H + L) (Li-COR, Cat# 926-68020, 1:20,000) were used for detection
using an Odyssey Classic imager (LI-COR).
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