Lysates for Western blotting were prepared using TCA extraction, as previously described [51 (link)]. Following SDS-PAGE and transfer to nitrocellulose, blots were probed with antibodies against PSTAIRE (P7962, Sigma), V5 (R96025, Invitrogen), MYC (clone 9E10, M5546, Sigma), Clb2 (y-180, Santa Cruz Biotechnology), Rad53 (ab104232, Abcam) or G6PDH (A9521, Sigma) as indicated.
A9521
Manufactured by Merck Group
Sourced in United States
A9521 is a laboratory equipment product offered by Merck Group. It serves as a general-purpose device for various laboratory applications. The core function of A9521 is to provide a stable and controlled environment for conducting experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ab104232, by Abcam (2 mentions)
Ab15083, by Abcam (2 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
P7962, by Merck Group (1 mentions)
Y 180, by Santa Cruz Biotechnology (1 mentions)
M5546, by Merck Group (1 mentions)
Optitran ba s 83 reinforced nc, by Cytiva (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Promega (1 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
Ab7766, by Abcam (1 mentions)
Ab8580, by Abcam (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Sc 2768, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (1 mentions)
Anti g6pdh, by Merck Group (1 mentions)
Sc 6773, by Santa Cruz Biotechnology (1 mentions)
Typhoon trio, by GE Healthcare (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
11 protocols using a9521
1
Protein Extraction and Western Blotting
2
Western Blot Analysis for Cas9 and G6PDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liquid cultures of each strain were grown to exponential phase in synthetic glucose medium or synthetic galactose medium without leucine, on order to maintain the Cas9 plasmid. Proteins were extracted on 2 x 108 cells in 200 μl Laemmli solution with 100 μl glass beads. Proteins were separated on a 10% acrylamide gel in standard conditions and blotted to a nitrocellulose membrane (Optitran BA-S 83 reinforced NC, Schleicher & Schuell). For Cas9 detection, a monoclonal HRP-conjugated mouse antibody was used (Abcam [7A9-3A3], ab202580, 1/1000 dilution). Note that blocking was achieved in 10 mM Tris-HCl pH8.0, NaCl 150 mM, 0.05% Tween 20, 3% dry milk (instead of the regular 5%). For G6PDH detection, the primary antibody was a polyclonal rabbit antibody (Sigma-Aldrich (A9521), 1/100 000 dilution). A secondary goat anti-rabbit antibody conjugated to horseradish peroxidase was used for detection of G6PDH (Thermo Scientific, 0.16 μg/ml final concentration). Quantification was performed using a ChemiDoc MP Imager (Bio-Rad) with the dedicated Image Lab software. The molecular weight marker used was the Precision Plus Protein Kaleidoscope marker (Bio-Rad).
3
Yeast Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from 1-2x108 yeast cells was done by suspending the cell pellet in 200 μL of NaOH 0.2M. After 5 minutes, samples were centrifuged and then mixed with 100 μL 2X SDS-PAGE loading buffer. They were incubated at 95°C for 5 minutes to completely extract proteins from cells. For the Western blot analysis [46 (link)], similar protein amounts were injected into SDS-PAGE acrylamide gels under reducing and denaturing conditions. Rabbit polyclonal antibodies against A190 and A135 RNA pol I subunits were assayed (diluted 1:5000) in this study, which were provided from O. Calvo and C. Fernández-Tornero. Glucose 6-phosphate dehydrogenase (G-6-PDH) was used as an internal control by re-incubating the same blots with a specific antibody (A9521 from Sigma Aldrich). Bound antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies (1:10000, Promega), followed by chemiluminescence detection with an ECL Prime Western blotting detection kit (GE Healthcare). At least three replicates of each sample were analyzed by using the ImageQuant LAS 4000 software (GE Healthcare).
4
TCA Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were prepared by TCA protein extraction method. Cells were resuspended in TCA buffer (1.85 M NaOH and 7.4% β-mercaptoethanol) for 10 min and then mixed with equal volume of 20% trichloroacetic acid to precipitate lysates. Cell lysates were dissolved in 0.1% NaOH and resolved using SDS-PAGE, then transferred onto PVDF membranes (Immobilon®-P, Millipore); membranes were then incubated with the indicated antibodies. The following antibodies were used in this study (dilutions used, source and catalog numbers): α-H2A S129-P (1:2000, Abcam, ab15083), α-H3K4me3 (1:10,000, Abcam, ab8580), α-H3K4me2 (1:5000 Abcam, ab7766), α-H3K4me1 (1:2000, Abcam, ab8895), α-HA (1:5000, Roche, 11867423001) and α-G6PDH (1:20,000, Sigma, A9521). Signals were detected using the ECL detection reagent (Millipore Immobilon™) with UVP BioSpectrum® imaging system. Uncropped blots can be found in Source data.
5
Quantitative Analysis of MAPK Activation Dynamics
6
Immunoblotting for Cytosolic Protein Purity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the cytosolic fractions to confirm the absence of any mitochondrial protein, was carried out using standard protocols, with antibodies specific for yeast cytosolic glucose-6-phosphate dehydrogenase (anti-G6PD, Sigma-Aldrich, A9521, 1:7500) and mitochondrial heat shock protein 60 (anti-Hsp60, Abcam, ab59458, 1:1000).
Immunoblotting after SDS-PAGE of chemically lysed yeast30 (link) was performed using standard protocols with antibodies specific for yeast glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, ThermoFisher, MA5-15738, 1:10.000) and HA (Sigma, H9658, 1:10000).
Immunoblotting after SDS-PAGE of chemically lysed yeast30 (link) was performed using standard protocols with antibodies specific for yeast glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, ThermoFisher, MA5-15738, 1:10.000) and HA (Sigma, H9658, 1:10000).
7
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were denatured in 1x SDS-sample buffer for 10 min at 70°C (for detection of Pmt2 and ER-GFP) or 3 min at 95°C (for detection of Hsp150), resolved on glycine polyacrylamide gels, and transferred to nitrocellulose membrane. Blots were incubated with primary antibodies anti-HA (from mouse; monoclonal; 1:10000; #MMS-101R; Covance), anti-Sec61 (from rabbit; polyclonal; 1:2500; gift from Karin Römisch), anti-GFP (from rabbit; polyclonal; 1:2500 #A6455; Thermo Fisher Scientific) or anti-G6PDH (from rabbit; polyclonal; 1:2500; #A9521; Sigma-Aldrich). Secondary antibodies horseradish peroxidase-conjugated anti-mouse (from rabbit; polyclonal; 1:5000; #A9044; Sigma-Aldrich) or anti-rabbit (from goat; polyclonal; 1:5000; #A6154; Sigma-Aldrich) were used. Protein-antibody complexes were visualized by enhanced chemiluminescence (Amersham ECL Detection Reagents; GE Healthcare).
8
Protein Isolation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated from the yeast cells by TCA (tri-chloroacetic acid) extraction, as described in [40 (link),51 (link)]. Briefly, about 4 × 108 cells in the exponentially growing phase were collected and resuspended in 200 μL of a 20% TCA solution. These cells in the TCA solution were lysed using the glass bead method. Proteins were then precipitated and resuspended in 200 μL of a Laemmli buffer and were neutralized with the addition of 100 μL of 1 M Tris, pH 8.8. The protein sample was boiled for 3 min, and 10 μg of the proteins was fractionated using SDS-PAGE in 4–12% gradient gels followed by Western blotting. The blot was probed in an Odyssey blocking buffer with the goat polyclonal anti-Rad53 (yC-19: sc6749, Santa Cruz Biotechnology, Dallas, TX, USA); the rabbit polyclonal anti-H2A phospho S129 antibody (ab15083, Abcam, Cambridge, UK); the rabbit polyclonal anti-HA (H6908, Sigma-Aldrich, St. Louis, MO, USA); or the rabbit polyclonal anti-G6PD (A9521, Sigma-Aldrich), followed by the secondary antibody LI-COR IRDye 800CW of the goat polyclonal anti-rabbit IgG (H+L) 926-32211 or the LI-COR IRDye 800CW donkey anti-goat IgG (H+L) 926-32214. Imaging was performed using the LI-COR Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
9
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were prepared by boiling the pelleted cells in radioimmunoprecipitation assay buffer, as described (41 (link)). The membranes were probed with 1:5000 mouse monoclonal anti-HA (16B12; Covance), 1:5000 rabbit polyclonal anti-FRB (from David Shore, University of Geneva), 1:5000 rabbit polyclonal anti-RAP1 (from David Shore, University of Geneva), 1:10,000 rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (A9521; Sigma-Aldrich), and 1:5000 mouse monoclonal anti-GFP antibodies (11814460001, Roche). Horseradish peroxidase–conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (A8275, Sigma-Aldrich) immunoglobulin G were used at 1:10,000 as secondary antibodies.
10
Yeast Strain Generation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast strains were grown under standard conditions, and gene deletion and tagging were performed as described previously (84 (link)). The list of yeast strains and plasmids used for various studies is provided in Table S1 and S2 , respectively. Plasmid transformations were performed as described previously (85 (link)). Primers used for deletion and tagging genes are listed in Table S3 . Genomic integration of the tagged and the deleted genes was confirmed by PCR. Antibodies used were as follows: anti-HA antibody, ChIP grade (ab9110, Abcam), anti-histone H4 antibody, ChIP grade (ab7311, Abcam), anti-histone H4Ac pan-acetyl antibody, ChIP grade (39243, Active Motif), H2A (1:5000; 39325, Active Motif), H2ASer129Ph (39271, Active Motif), anti-Rad53 (ab104232, Abcam), G6PDH (1:100,000; A9521, Sigma-Aldrich), and rabbit (Amersham NA934; donkey anti-rabbit) and mouse (Amersham NA931; sheep anti-mouse) secondary antibodies were used at 1:10,000.
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!