Sc 7392

Manufactured by Merck Group

Sc-7392 is a laboratory instrument designed for spectrophotometric analysis. It is capable of measuring the absorbance or transmittance of light through samples across a range of wavelengths.

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Lab products found in correlation

Anti ha antibody, by Santa Cruz Biotechnology (2 mentions) Flag m2 sepharose beads, by Merck Group (1 mentions) A1949, by Merck Group (1 mentions) Anti tubulin monoclonal antibody, by Merck Group (1 mentions) Deltavision spectris microscope, by Cytiva (1 mentions) Crl1729, by American Type Culture Collection (1 mentions) Sc 40, by Santa Cruz Biotechnology (1 mentions) Protein g sepharose, by Santa Cruz Biotechnology (1 mentions) Anti sp1 antibody, by Merck Group (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

4 protocols using sc 7392

Mitochondrial Protein Purification and Identification

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Mitochondria (250 µg of protein) were lysed for 5 min on ice in 250 µl buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1 mM DTT, 10 % v v⁻¹ glycerol, 0.2% v v⁻¹ Triton-X100) supplemented with 2 mM phenylmethylsulphonylfluoride. Membrane debris was removed by centrifugation (12000×g, 15 min, 4 °C) and the mitochondrial lysate was incubated with 30 µl FLAG-M2 Sepharose beads (Sigma Aldrich) for 1 h on a rotary shaker at 4 °C. Beads were pelleted by centrifugation (1000×g, 1 min, 4 °C) and washed 3 times with 500 µL buffer. Bound proteins were subjected to SDS polyacrylamide gel electrophoresis and identified by immunostaining using specific antibodies (α-Nfs1, α-Isu1, and α-porin: self-raised (1:5000; rabbit), α-HA: Santa Cruz (sc-7392) (1:15,000; mouse); α-FLAG: SIGMA (011M4789) (1:20,000; rabbit); secondary α-mouse: Bio-Rad (L1706516) (1:1000); secondary α-rabbit: Sigma-Aldrich (A1949) (1:1000)).

Boniecki M.T., Freibert S.A., Mühlenhoff U., Lill R, & Cygler M. (2017). Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex. Nature Communications, 8, 1287.

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Detailed Western Blot and Immunofluorescence Protocols

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Western analysis was carried out as described previously [13] (link). Production of anti-SUMO and anti-eIF4GI (against the KRERK epitope) antisera has been described elsewhere [41] (link), [42] (link), anti-myc antibodies for immunofluorescence were purified from cell supernatant (cell line CRL1729, from ATCC) using protein G-sepharose or were from Santa Cruz (sc-40), anti-HA antisera were from Santa Cruz (sc-7392) and monoclonal anti-tubulin antibodies were from Sigma (T5168). Immunofluorescence was undertaken as described in Moreno et al. [43] (link). Cells were observed using an Applied Precision Deltavision Spectris microscope using deconvolution software.

Jongjitwimol J., Feng M., Zhou L., Wilkinson O., Small L., Baldock R., Taylor D.L., Smith D., Bowler L.D., Morley S.J, & Watts F.Z. (2014). The S. pombe Translation Initiation Factor eIF4G Is Sumoylated and Associates with the SUMO Protease Ulp2. PLoS ONE, 9(5), e94182.

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Plasmid Construction and Reagents

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The PACS1-HA plasmid was provided by Dr. Gary Thomas (University of Pittsburg). The PACS1-HA sequences were inserted into the MLV-based vector pBabe (provided by Dr. Richard Sutton, Yale University). The pCMVGagPol-RRE and pCMV-RevFlag plasmids were provided by Dr. Marie-Louise Hammarskjöld (University of Virginia). The Flag-Vpr plasmid was constructed by PCR amplification of the NL4-3 Vpr gene and insertion into a CMV-Flag expression plasmid. The influenza virus Flag-NS1 plasmid contains a mutation in the NS1 binding site to CPSF30 to increase NS1 protein expression (Golebiewski, Liu et al., 2011 (link)). The pHMRLuc (Renilla) and Rem-GFP (Green Fluorescent Protein) plasmids were provided by Dr. Jaquelin Dudley (University of Texas). PACS1 (sc-106348) and control (sc 37007) small interfering RNAs (siRNAs) were from Santa Cruz Biotechnology. PACS1 antiserum (ab56072) was from Abcam; anti-HA antibody was from Santa Cruz Biotechnology (sc-7392); anti-CRM1 antibody (ST1100) and Flag antibody (F1804) were from Millipore and Sigma-Aldrich, respectively.

Liu H., Hu P.W., Budhiraja S., Misra A., Couturier J., Lloyd R.E., Lewis D.E., Kimata J.T, & Rice A.P. (2019). PACS1 is an HIV-1 cofactor that functions in Rev-mediated nuclear export of viral RNA. Virology, 540, 88-96.

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ChIP-seq protocol for K562 cells

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K562 cells in two T75 flasks were treated with 4OHT at 100 nM for indicated times, and ChIP was performed as described previously (Hasegawa and Struhl 2019) , except for a change in the sonication program (Misonix 3000 sonicator, level 2, ON 30 sec, OFF 30 sec, total sonication time 8 min). The protein concentration of the lysates was measured by BCA Protein Assay Kit (Thermo Scientific) and 1.7 µg total protein was used for the immunoprecipitation step. Yeast chromatin were prepared from HA-TBP expressing strain as previously described (Hasegawa and Struhl 2019 ) and added to the lysates at a constant ratio (3% of total protein amount in the K562 cell lysates) as a spike-in control. For immunoprecipitation, 0.3 µg of anti-HA antibody (Santa Cruz #sc-7392) or 3 µg of anti-SP1 antibody (Millipore #07-645) were used. Primers for quantitative PCR are listed in Table S3.

Hasegawa Y., & Struhl K. (2020). Different SP1 binding dynamics at individual genomic loci in human cells.

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