mPFC tissue was collected as described above. The Epiquik Total Histone Extraction Kit (OP-0006, EpiGentek, Farmingdale, NY) was used to isolate the histone fraction. A BCA assay was used to determine protein concentration. After boiling in laemmli buffer for 5 minutes, 15 μg of protein were loaded on a 15% SDS-PAGE gel. After electrophoresis, blots were transferred to pore size 0.20 μm polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat milk and separately probed with the following primary antibodies: H3K9ac (RRID:AB_297491; Abcam, ab10812, 1:5000, Cambridge, UK), H3K18ac (RRID:AB_298692; Abcam, ab1191, 1:5000), H3K27ac (RRID:AB_2118291; Abcam, ab4729, 1:5000), H3K27me3 (RRID:AB_305237; Abcam, ab6002, 1:2000), and total histone H3 (RRID:AB_10001790; Novus, NB500-171, 1:100,000) was used as a loading control. Blots were incubated with horseradish peroxidase-coupled anti-rabbit or anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA), and proteins were visualized using enhanced chemiluminescence (ECL Plus, Amersham Biosciences, Piscataway, NJ). Protein expression for each histone modification was evaluated by densitometry using Image-J software. Samples from each animal were run at least 4 times to minimize interblot variance.
Nb500 171
Manufactured by Novus Biologicals
Sourced in United Kingdom
The NB500-171 is a laboratory equipment product offered by Novus Biologicals. It is a device designed for a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Epiquik total histone extraction kit, by Epigentek (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab1191, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
Ab10812, by Abcam (1 mentions)
Ab5535, by Merck Group (1 mentions)
Ab5603, by Merck Group (1 mentions)
Novex ecl chemi substrate, by Thermo Fisher Scientific (1 mentions)
Ab32064, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
Ab24610, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
Nb100 122, by Novus Biologicals (1 mentions)
Nb100 110, by Novus Biologicals (1 mentions)
Nb100 479, by Novus Biologicals (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
5 protocols using nb500 171
1
Histone Modification Analysis in mPFC
2
Immunoblot Analysis of Protein Expression
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Immunoblots were performed following standard procedures24 (link). Specific antibodies against HDAC6 (7558 S Cell Signalling), acetyl-α-tubulin (ab24610, Abcam), α-tubulin (ab52866, Abcam), acetyl-histone H3-lys9 (9649, Cell Signaling), histone H3 (NB500-171, Novus), PARP (ab32064, Abcam), BMI-1 (05-637, Millipore), SOX2 (AB5603, Millipore), SOX9 (AB5535, Millipore), and β-actin (A5441, Sigma) were used in the study. For secondary antibodies, horseradish peroxidase (HRP)-linked anti-rabbit (7074S, Cell Signalling) or anti-mouse (7076S, Cell Signalling) were used. Detection was performed by chemiluminiscence using NOVEX ECL Chemi Substrate (ThermoFisher).
3
Chromatin immunoprecipitation of hypoxia-responsive transcription factors
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Hep3B and SMMC-7721 cells were incubated at 20% or 1% O2 for 16h, cross-linked in 3.7% formaldehyde for 15 minutes, quenched in 0.125 M glycine for 5 minutes, and lysed with SDS lysis buffer. Chromatin was sheared by sonication, and lysates were precleared with salmon perm DNA/protein A agarose slurry (Millipore) for 1 hour and incubated with antibodies against IgG (2 μg/sample, NBP2-36463, Novus), HIF-1α (2 μg/sample, NB100-479, Novus), HIF-1β (2 μg/sample, NB100-110, Novus), HIF-2α (2 μg/sample, NB100-122, Novus), H3K9ac (2 μg/sample, NB21-1017, Novus), H3 (2 μg/sample, NB500-171, Novus) or HDAC3 (2 μg/sample, NB100-1669, Novus) in the presence of protein A-agarose beads overnight. After serial washing of the agarose beads with low-salt, high-salt, and LiCl buffers, DNA was eluted in 1% SDS with 0.1M NaHCO3, and cross links were reversed by addition of 0.2M NaCl. DNA was purified by phenol-chloroform extraction and ethanol precipitation, and analyzed by qPCR. The primers were shown as below. MIR627-HRE-1: Forward: 5'-GGATTAATTCCCCCATTGCT-3', Reverse: 5'-GTACCCCCTCATCCATGTTG-3. MIR627-HRE-2: Forward: 5'-CTCCCAAAGTGCTGGGATTA-3', Reverse: 5'-CTTCCCTTCGTTGGGTAACA-3'. HDAC3-HRE: Forward: 5'-CTTCCCAAATGCCTTCACC-3', Reverse: 5'-ACCGGATAGACCAGTGGACA-3'. Primers used for MIR627 gene promoter PCR: Forward: 5'- TCCTGCCAGCAGTGTATGAG-3', Reverse: 5'-ACCAGGTATCCAACCTCAGC-3'.
4
Protein Extraction from Yeast Cells
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Exponentially growing cells inoculated in YES were arrested with 0.1% sodium azide. 1x108 cells were collected to perform the following protein extraction. The cell pellet was suspended and washed in 1 ml stop buffer (50 mM NaF, 10 mM NaN3 in PBS 1X) and then washed in 1 ml of 20% trichloroacetic acid (TCA). Pelleted cells were resuspended in 200 μl 20% TCA and glass beads (Sigma G8772) were added to each tube. The cell walls were mechanically broken using a FastPrep-24 bead beating homogenizer (MP-Biomedicals) with the following program: 6000 rpm, 3 rounds of 30 sec ON with a one-minute interval between each round on ice. 400 μl 5% TCA was added to the cell lysate and cell lysate was recovered without the beads in a new tube. The cell lysate was spun at 13k rpm for 5 min and the pellet was resuspended in 200 μl TCA buffer (1x SDS loading buffer, 0.2M Tris-HCl pH 8). The samples were denatured by boiling at 95°C for 5 min followed by a brief centrifugation prior to western blot analysis. Proteins were resolved by 4–20% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was probed with anti-HA (1:2000, Sigma H6908) or anti-H3 (1:5000, Novus NB500-171) antibodies and the proteins were revealed by chemiluminescence with horseradish-peroxidase-conjugated sheep anti-rabbit IgG (cytiva).
5
Protein Extraction and Immunoblotting Protocol
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For protein extract preparation, cells were washed twice with ice-cold phosphate buffered saline (PBS), resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 100 mg/mL PMSF). The supernatant, obtained by centrifugation at 16000 g for 15 minutes, was transferred into a new tube and used to determine protein concentrations by Bradford assay. Cell lysates were resolved by SDS/PAGE and transferred to PVDF membranes (GE, Healthcare). The membranes were blocked in TTBS (TBS with 0.1% Tween 20) containing 5% milk. Primary antibody incubations were performed in TTBS with either 5% BSA, overnight at 4 °C. After washing, the membranes were incubated with the appropriate secondary peroxidase conjugated antibody for 1 hour in TTBS. Following antibodies were used for IB: anti-DHX9 (sc-137,232), anti-GAPDH (sc-365,062), anti-AR (sc-7305), anti-H3 (Novus, NB500–171). Proteins were visualized by chemiluminescence detection system (Clarity Western ECL Substrate, #1705061) and quantification analysis was performed using Image J Software.
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