All ChIP experiments were conducted in ΔSnf1 LS41 strain with pGADT7-Snf1-6xmyc complemented back in. Cultures were grown in 2% glucose to mid-log phase and then induced for 3 h in 2% galactose before treating with formaldehyde. Solubilized chromatin was split equally among three clean tubes to which 2 μg Snf1 antibody (sc-15621, Santa Cruz Biotechnologies), LexA antibody (sc-1725, Santa Cruz Biotechnologies), or control IgG (sc-2027, Santa Cruz Biotechnologies) was added. Immunoprecipitated samples were immobilized to magnetic Dynabeads Protein G (Life Technologies) and washed. Samples were eluted at 65 °C for 30 min, the eluate transferred to a new tube, and the formaldehyde cross-links were reversed overnight in a 65 °C water bath. Samples were purified using a Qiagen PCR clean up kit prior to qPCR analysis. qPCR reactions were set up in triplicate for each sample using Promega GoTaq qPCR Master Mix reagents. All samples were run on an Applied Biosystems StepOne Plus, and a minimum of three independent experiments were conducted to generate average % input values for each immunoprecipitation condition. Detailed experimental procedure and data analysis can be found in the Supplementary Methods .
Lexa antibody
Manufactured by Santa Cruz Biotechnology
The LexA antibody is a laboratory tool used for detecting and analyzing the LexA protein in biological samples. LexA is a transcriptional repressor that plays a key role in the SOS response, a cellular process that is activated in response to DNA damage. The LexA antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify the presence of LexA in cells or tissues.
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Lab products found in correlation
Pcr cleanup kit, by Qiagen (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Control igg, by Santa Cruz Biotechnology (1 mentions)
Gotaq qpcr master mix reagent, by Promega (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Superblock t20 blocking buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
2 protocols using lexa antibody
1
Snf1-Mediated Chromatin Immunoprecipitation
2
Western Blot Assay for RNA Polymerase
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Cells were diluted 1,000-fold from overnight cultures and grown to an OD600 of 0.2 in 2 ml of medium before the addition of 80 µg ml–1 Rif for 1 h. Cells were then collected, resuspended in lysis buffer (10 mM Tris-HCl pH 7.5, 4% SDS), mixed with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen), and boiled at 95 °C for 10 min before loading on a NuPAGE 4–12% Bis-Tris precast gel (Thermo) for electrophoresis at 200 V for 40 min. Gel was then semidry transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) and incubated with the specified antibodies at 4 °C overnight in SuperBlock T20 blocking buffer (Thermo) before development the following day. Antibodies used were E. coli RNAP beta Monoclonal Antibody clone 8RB13 mouse mAB at 1:2,000 (663905; Biolegend) and LexA Antibody (E-7) at 1:200 (sc-365999; Santa Cruz). Gel intensity was quantified using GelQuant.NET.
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