Criterion XT Bis-Tris Gels (4–12% polyacrylamide, BioRad: Hercules, CA, USA), XT MES Buffer (BioRad) and Trans-Blot® Turbo™ Midi Transfer Packs were used for electrophoresis and immunoblotting (n = 2–4) on PVDF membranes. Phospho-AMPKα (Thr172; 2531) and Phospho-AKT (Ser473; 4060) antibodies were sourced from Cell Signaling Technology: Danvers, MA, USA. Images were obtained with a ChemiDoc + XRS and immunoblots were quantified with ImageLab analysis software (BioRad). IL-6 was quantified in the conditioned media using a commercially available mouse IL-6 ELISA kit (n = 10; 431301, BioLegend: San Diego, CA, USA).
Xt mes buffer
Manufactured by Bio-Rad
Sourced in United States
The XT MES Buffer is a laboratory reagent used for electrophoresis procedures. It is a buffered solution designed to maintain a specific pH environment during the separation and analysis of macromolecules, such as proteins and nucleic acids, in an electrophoresis system.
Automatically generated - may contain errors
Lab products found in correlation
Criterion xt bis tris gel, by Bio-Rad (4 mentions)
Pmsf protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Supersignal west pico and femto chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Image lab software v5, by Bio-Rad (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Mouse il 6 elisa kit, by BioLegend (1 mentions)
Image lab analysis software, by Bio-Rad (1 mentions)
Phospho akt ser473 4060, by Cell Signaling Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Bio-Rad (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Total p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Phospho histone h3 ser10, by Merck Group (1 mentions)
Total histone h3, by Cell Signaling Technology (1 mentions)
Xt mops buffer, by Bio-Rad (1 mentions)
Total bad, by Cell Signaling Technology (1 mentions)
Poly adpribose polymerase, by BD (1 mentions)
6 protocols using xt mes buffer
1
Quantifying AMPK and AKT Phosphorylation
2
CLL Cell Protein Expression Analysis
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Cells were untreated or treated with DMSO or AZD1208. CLL cells were harvested after treatment, centrifuged, and washed twice with PBS. Cells were lysed using one tablet of Complete Mini Protease Inhibitor Cocktail (Roche) in 10 mL of 1 x radioimmunoprecipitation assay buffer (Bio-Rad; Hercules, CA, USA) or 1 x Lysis Buffer (Cell Signaling). The lysate protein content was measured using a DC protein assay kit (Bio-Rad), according to the manufacturer’s instructions. Protein samples were electrophoresed on Criterion XT Bis-Tris gels using either XT MOPS buffer or XT MES buffer (Bio-Rad) and were transferred either to nitrocellulose or PVDF membranes. Primary antibodies were purchased from the following sources: BCL-2 (Dako; Carpinteria, CA, USA); MCL-1, BCL-XL, and c-MYC (clone C33) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); phospho-4E-binding protein 1 (4E-BP1) (Thr37/46), phospho-4E-BP1 (Ser65), total 4E-BP1, phospho-p70 S6 kinase (Thr389), total p70 S6 kinase, glyceraldehyde 3-phosphate dehydrogenase, total Bad, total histone H3, and LC3A/B (Cell Signaling Technology; Danvers, MA, USA); phospho c-MYC-1 (Abcam; Cambridge, MA, USA); phospho-histone H3 (Ser 10) (EMD Millipore; Billerica, MA, USA); and poly ADP ribose polymerase (BD Pharmingen); p62 (Enzo Life Sciences, Farmingdale, NY).
3
Immunoblotting protocol for protein analysis
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Immunoblotting analysis was performed as previously reported (Vanhulle et al., 2022 (link)). Cells were collected and lysed in ice-cold NP-40 lysis buffer (50 mM Tris-HCL (pH 8.0), 150 mM NaCl, and 1% Nonidet P-40) supplemented with cOmplete Protease Inhibitor (Roche) and PMSF Protease Inhibitor (100 mM in dry isopropanol, Thermo Fisher Scientific). Cell lysates were centrifuged at 17,000 g for 10 min at 4°C to pellet nuclei and debris. For SDS gel electrophoresis, supernatant samples were boiled in reducing 2x Laemmli sample buffer (120 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM dithiothreitol, and 0.02% bromophenol blue). Equal volumes of lysate were run on Criterion XT Bis-Tris gels (4–12%; Bio-Rad) at 170 V for 55 min using 1x XT-MES buffer (Bio-Rad), transferred to nitrocellulose membranes using the BioRad Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked for 1 h with 5% non-fat dried milk in TBS-T (20 mM Tris-HCL (pH 7.6), 137 mM NaCl, and 0.05% Tween-20). After overnight incubation with primary antibody at 4°C, membranes were washed and incubated for 1h with secondary antibody. β-actin was used as a loading control. SuperSignal West Pico and Femto chemiluminescence reagent (Thermo Fisher Scientific) was used for detection with a ChemiDoc MP system (Bio-Rad). Signal intensities were quantified with Image Lab software v5.0 (Bio-Rad).
4
Western Blot Analysis of Protein Lysates
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Cells were collected and lysed in ice-cold Nonidet P-40 lysis buffer (50 mM Tris-HCL (pH 8.0), 150 nM NaCl, and 1% Nonidet P-40) supplemented with 100x cOmplete Protease Inhibitor (Roche) and 250x PMSF Protease Inhibitor (100 mM in dry isopropanol, Thermo Fisher Scientific). Cell lysates were centrifuged at 17,000 g for 10 min at 4 °C to pellet nuclei and debris. For SDS gel electrophoresis, supernatant samples were boiled in reducing 2x Laemmli sample buffer (120 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM dithiothreitol, and 0.02% bromophenol blue). Equal volumes of lysate were run on Criterion XT Bis-Tris gels (4–12%; Bio-Rad) at 170 V for 55 min using 1x XT-MES buffer (Bio-Rad), transferred to nitrocellulose membranes using the BioRad Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked for 1 h with 5% non-fat dried milk in TBS-T (20 mM Tris-HCL (pH 7.6), 137 mM NaCl, and 0.05% Tween-20). After overnight incubation with primary antibody at 4 °C, membranes were washed and incubated for 1h with secondary antibody. Clathrin and β-actin were used as loading control. SuperSignal West Pico and Femto chemiluminescence reagent (Thermo Fisher scientific) was used for detection with a ChemiDoc MP system (Bio-Rad). Signal intensities were quantified with Image Lab software v5.0 (Bio-Rad).
5
SDS-PAGE Protein Separation Protocol
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Fractions from SmE protein purification were run on a 4–12% Criterion XT BisTris gel (Bio-Rad, 3450123) in MES XT buffer (Bio-Rad, 1610789) at 180 V for 60 min, alongside Precision Plus All Blue Protein Standard (Bio-Rad, 1610373). Digested proteins were separated on a 4–12% Criterion XT BisTris gel (Bio-Rad, 3450123) in MOPS XT buffer (Bio-Rad, 1610788) at 180 V for 60 min, alongside Blue Easy Protein Ladder (NIPPON Genetics, MWP06). All protein gels were imaged on a LiCOR Odyssey instrument following a 30 min incubation with Aquastain (Bulldog Bio, AS001000).
6
Comparative Analysis of Anti-FLAG-M2 Antibodies
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To
test the binding of our recombinant anti-FLAG-M2 to the FLAG-tag epitope,
compared to the commercially available anti-FLAG-M2 (Sigma-Aldrich),
we used both antibodies to probe Western blots of a FLAG-tagged protein
in parallel. Purified Rabies virus glycoprotein ectodomain (SAD B19
strain, UNIPROT residues 20–450) with or without a C-terminal
FLAG-tag followed by a foldon trimerization domain and an octahistidine
tag was heated to 95 °C in XT sample buffer (Bio-Rad) for 5 min.
Samples were run twice on a Criterion XT 4–12% polyacrylamide
gel (Bio-Rad) in MES XT buffer (Bio-Rad) before Western blot transfer
to a nitrocellulose membrane in Tris–glycine buffer (Bio-Rad)
with 20% methanol. The membrane was blocked with 5% (w/v) dry nonfat
milk in phosphate-buffered saline (PBS) overnight at 4 °C. The
membrane was cut into two (one half for the commercial and one half
for the recombinant anti-FLAG-M2), and each half was probed with either
commercial (Sigma-Aldrich) or recombinant anti-FLAG-M2 at 1 μg/mL
in PBS for 45 min. After washing three times with PBST (PBS with 0.1%
v/v Tween20), polyclonal goat antimouse fused to horseradish peroxidase
(HRP) was used to detect binding of anti-FLAG-M2 to the FLAG-tagged
protein for both membranes. The membranes were washed three more times
with PBST before applying enhanced chemiluminescence (ECL; Pierce)
reagent to image the blots in parallel.
test the binding of our recombinant anti-FLAG-M2 to the FLAG-tag epitope,
compared to the commercially available anti-FLAG-M2 (Sigma-Aldrich),
we used both antibodies to probe Western blots of a FLAG-tagged protein
in parallel. Purified Rabies virus glycoprotein ectodomain (SAD B19
strain, UNIPROT residues 20–450) with or without a C-terminal
FLAG-tag followed by a foldon trimerization domain and an octahistidine
tag was heated to 95 °C in XT sample buffer (Bio-Rad) for 5 min.
Samples were run twice on a Criterion XT 4–12% polyacrylamide
gel (Bio-Rad) in MES XT buffer (Bio-Rad) before Western blot transfer
to a nitrocellulose membrane in Tris–glycine buffer (Bio-Rad)
with 20% methanol. The membrane was blocked with 5% (w/v) dry nonfat
milk in phosphate-buffered saline (PBS) overnight at 4 °C. The
membrane was cut into two (one half for the commercial and one half
for the recombinant anti-FLAG-M2), and each half was probed with either
commercial (Sigma-Aldrich) or recombinant anti-FLAG-M2 at 1 μg/mL
in PBS for 45 min. After washing three times with PBST (PBS with 0.1%
v/v Tween20), polyclonal goat antimouse fused to horseradish peroxidase
(HRP) was used to detect binding of anti-FLAG-M2 to the FLAG-tagged
protein for both membranes. The membranes were washed three more times
with PBST before applying enhanced chemiluminescence (ECL; Pierce)
reagent to image the blots in parallel.
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