Application
ChIP
Sample
Human Cell lysate - whole cell (HeLa)
Specification
HeLa
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step
Real-time PCR
Positive control
Positive control primers for ALDH12 gene transcribed by mtRPol
Negative control
Negative control PCR primers used for an intergenic chromosome 7 sequence.
Other product details
Concentration
0.12 µg/µg chromatin
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 20mM TrispH 8,150 mM NaCl, 1.5 mM EDTA
Additional data
Additional Notes
General ChIP notes
Extracts prepared in ChIP lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.l% SDS) + protease inhibitors (PMSF, Roche Protease Inhibitor Cocktail). Chromatin sheared 6 x 30 sec pulses to average fragment length <1 kb. Extracts clarified by centrifugation.
3 ug antibody added to 25 ug precleared chromatin extract diluted 1/10 with dilution buffer (20 mM Tris pH 8, 150 mM NaCl, 1.5 mM EDTA, 1% Triton X-100) + protease inhibitors. After overnight incubation with antibody, 50 uL Protein G beads added for 5 h. Beads washed 3x low salt wash buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), 1x high salt wash buffer (20 mM Tris pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS). Protein eluted for 15 min in elution buffer (100 mM NaHCO3, 1% SDS) at room temperature.
5 uL Proteinase K 20 mg/mL added to eluted protein, then incubated in water bath at 65C overnight.
DETECTION METHOD: ABI 7900 Real-time PCR instrument Promega GoTaq qPCR Master Mix + CXR (equivalent to SYBR Green + ROX).
Extracts prepared in ChIP lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.l% SDS) + protease inhibitors (PMSF, Roche Protease Inhibitor Cocktail). Chromatin sheared 6 x 30 sec pulses to average fragment length <1 kb. Extracts clarified by centrifugation.
3 ug antibody added to 25 ug precleared chromatin extract diluted 1/10 with dilution buffer (20 mM Tris pH 8, 150 mM NaCl, 1.5 mM EDTA, 1% Triton X-100) + protease inhibitors. After overnight incubation with antibody, 50 uL Protein G beads added for 5 h. Beads washed 3x low salt wash buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), 1x high salt wash buffer (20 mM Tris pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS). Protein eluted for 15 min in elution buffer (100 mM NaHCO3, 1% SDS) at room temperature.
5 uL Proteinase K 20 mg/mL added to eluted protein, then incubated in water bath at 65C overnight.
DETECTION METHOD: ABI 7900 Real-time PCR instrument Promega GoTaq qPCR Master Mix + CXR (equivalent to SYBR Green + ROX).
Abcam response
Our Scientific Support team is currently investigating this negative Abreview, and we will resolve the issue in accordance with our Abpromise.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
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投稿 Feb 28 2012