Sample preparation for western blot analysis was performed as in Denoncin et al.,83 (link) starting from 3 mL in the case of cleared B. bacteriovorus lysates. Sample were loaded on NuPage Bis-Tris SDS precast polyacrylamide gels and ran at 190 V for 50 minutes in NuPAGE MES SDS running buffer. Western blotting was performed using standard procedures with the following primary antibodies: JL-8 monoclonal antibody (Takara) for GFP variants, YFP and CFP; polyclonal mCherry antibody (product # PA5-34974, Thermo Fisher) for mCherry. Signal from antibody binding was visualized by detecting chemiluminescence from the reaction of horseradish peroxidase with luminol and chemiluminescence was imaged with an Image Quant LAS 500 camera (GE Healthcare). Goat anti-mouse IgG-peroxidase antibody (Sigma) was used as a secondary antibody for JL-8. Goat anti-rabbit IgG-peroxidase antibody (Sigma) was used as a secondary antibody for mCherry. Antibodies were diluted following manufacturer’s recommendations. Figures were prepared using ImageJ and assembled and annotated in Adobe Illustrator.
Imagequant las 500 camera
Manufactured by GE Healthcare
The ImageQuant LAS 500 camera is a high-performance imaging system designed for life science research applications. It is capable of capturing and analyzing images of various types of samples, including gels, blots, and other biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds running buffer, by Bio-Rad (2 mentions)
Trans blot turbo rta mini pvdf transfer kit, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Mcherry antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg peroxidase antibody, by Merck Group (1 mentions)
Goat anti rabbit igg peroxidase antibody, by Merck Group (1 mentions)
Mini protean tgxtm precast gel, by Bio-Rad (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Supersignal west pico plus kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Nupage 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Ecl plus immunoblotting chemiluminescence system, by GE Healthcare (1 mentions)
5 protocols using imagequant las 500 camera
1
Western Blot Analysis of Fluorescent Proteins
2
Western Blot Analysis of HSP90 and LDHA
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Homogeneous tumor powder was lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount was measured with a PierceTM BCA protein Assay Kit (Thermo Scientific). Equal amounts of proteins were loaded onto 4–15% Mini-PROTEAN TGXTM Precast Gels (Bio-Rad). Following electrophoresis in 1× Tris/glycine/SDS running Buffer (Bio-Rad), proteins were transferred to PVDF membranes using the Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad) according to the vendor’s instructions. Non-specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1* Tris-Buffered Saline, 0.1% Tween 20, Bio-Rad) at room temperature for 1 h.
Membranes were incubated with primary anti-HSP90, anti-LDHA (Cell Signaling, #2021S, dilution 1:1000), in tTBS-BSA 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR) in tTBS-BSA 1% at room temperature for 1 h. Detection was performed using the SuperSignalTM West Pico Plus Kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No Background correction was performed.
Membranes were incubated with primary anti-HSP90, anti-LDHA (Cell Signaling, #2021S, dilution 1:1000), in tTBS-BSA 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR) in tTBS-BSA 1% at room temperature for 1 h. Detection was performed using the SuperSignalTM West Pico Plus Kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No Background correction was performed.
3
Western Blot Analysis of Metabolic Proteins
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Adherent A375 and A375R cells were lysed in RIPA buffer (Thermo Scientific) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount in whole cell lysates was measured with a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were loaded onto 4%‐15% Mini‐PROTEAN® TGX™ Precast Gels (Bio‐Rad). Following electrophoresis in 1× Tris/glycine/SDS running buffer (Bio‐Rad), proteins were transferred to PVDF membranes using the Trans‐Blot® Turbo™ RTA Mini PVDF Transfer Kit (Bio‐Rad) according to the vendor's instructions. Non‐specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1× Tris‐Buffered Saline, 0.1% Tween 20, Bio‐Rad) at room temperature for 1 hour.
Membranes were incubated with primary anti‐HSP90, anti‐LDHA, anti–β‐actin, anti‐HSP90, anti–c‐MYC, (Cell Signaling), anti‐MCT1, anti‐MCT4, anti‐GLUT1 (Abcam) antibodies in tTBS‐BSA 5% at 4°C overnight, followed by incubation with anti‐rabbit or anti‐mouse secondary antibodies (Jackson IR) in tTBS‐BSA 1% at room temperature for 1 hour. Detection was performed using the SuperSignal™ West Pico Plus kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No background correction was performed.
Membranes were incubated with primary anti‐HSP90, anti‐LDHA, anti–β‐actin, anti‐HSP90, anti–c‐MYC, (Cell Signaling), anti‐MCT1, anti‐MCT4, anti‐GLUT1 (Abcam) antibodies in tTBS‐BSA 5% at 4°C overnight, followed by incubation with anti‐rabbit or anti‐mouse secondary antibodies (Jackson IR) in tTBS‐BSA 1% at room temperature for 1 hour. Detection was performed using the SuperSignal™ West Pico Plus kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No background correction was performed.
4
Immunoblotting for CnoX Protein
5
Western Blot Analysis of Protein Expression
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Cells transfected with shRNA were lysed in NP-40 lysis buffer (25 mM Tris, pH 7–8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100), and protein concentration in the cell lysate was quantified using a Bradford protein assay (Bio- Rad), as previously described30 (link). Protein samples were incubated at 100 °C for 5 min and electrophoresed on 10% SDS–polyacrylamide gels. Proteins were transferred to PVDF membranes (GE Healthcare Life Sciences). Membranes were blocked in 5% non-fat milk in Tris- buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Membranes were then incubated at 4 °C with following primary antibody rabbit anti-EHD1 (24657-1-AP, Proteintech), mouse anti-α-tubulin (T5168, Sigma) diluted in 5% non-fat milk overnight (1:500 and 1:10,000, respectively) then washed several times with TBS-T. After 1-h incubation with horseradish peroxidase-conjugated with respective IgG secondary antibody (1:10,000) (GE Healthcare Life Sciences), membranes were washed with TBS-T and protein bands on the membrane were detected using an ECL-Plus immunoblotting chemiluminescence system (GE Healthcare Life Sciences). Membranes were imaged using ImageQuant LAS 500 camera (GE Healthcare Life Sciences), as previously described30 (link).
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