Proteomics & Protien Expression

Fine Mapping of Genomic Targets

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Selected Protocol from "Protein-Protein Interactions", 2nd Edition, E. Golemis, P. Adam.

Fine Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells

Achim Breiling and Valerio Orlando
Dulbecco Telethon Institute, Institute of Genetics and Biophysics, CNR, 80131 Naples, Italy


ABSTRACT
The aim of this technique is to analyze interactions of proteins or protein complexes with chromosomal DNA.

The following protocol has been established for the cultured cell line Schneider 2 (S2) from Drosophila melanogaster. If other tissue is used, certain steps of the protocol may need to be optimized, although the basic rationale would be the same. The main difference is likely to be in the cross-linking step. Cells growing in suspension can be easily cross-linked by adding a concentrated stock of the cross-linking buffer directly to the growth medium (see below). The same procedure is used for yeast (Strahl-Bohlsinger et al. 1997). Adherent mammalian cells are also cross-linked by adding a concentrated stock of the cross-linking buffer to the medium. After cross-linking and quenching with glycine, the cells are scraped and washed off the culture dishes with PBS and pooled. Then cells are lysed according to the protocol below (see Fig. 2). When using more compact material, such as embryos or imaginal discs, cross-linking conditions are more vigorous and might include treatments with detergents or polar solvents, which allow the formaldehyde to better penetrate the sample. For additional protocols, see Cao et al. (2002; imaginal discs from D. melanogaster), Orlando et al. (1998; embryos from D. melanogaster), and Chua et al. (2004; tobacco shoots). For further specialized protocols, see also the Web sites listed after the reference section.

 

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Products Available for this Protocol
Protocol Material Description Product #  Product Name Add to Cart
General Materials      
Glycine (powder) G8790 Glycine, >98.5%, from non-animal source, cell culture tested, meets EP, JP & USP testing specifications
PBS (phosphate-buffered saline [pH 7.4]) P5368 Phosphate buffered saline
SDS, 10% L4390 Sodium dodecyl sulfate
Proteinase K P4850 Proteinase K from Tritirachium album
Phenol/chloroform/isoamyl alcohol (25:24:1) P2069 Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA
Chloroform/isoamyl alcohol (24:1) C0549 Chloroform:Isoamyl alcohol 24:1
Sodium acetate, 3 m (pH 5.2) S7545 Sodium acetate
Glycogen (5 mg/ml) G0885 Glycogen from bovine liver
Ethanol, 100% and 70% E7148 Ethanol
Fixation solution      
11% formaldehyde F8775 Formaldehyde solution
100 mm NaCl S3014 Sodium chloride
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
0.5 mm EGTA E3889 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
50 mm HEPES H4034 HEPES
100 mm PMSF (phenylmethylsulfonyl fluoride) P7626 Phenylmethanesulfonyl fluoride
Cell lysis buffer      
5 mm PIPES (pH 8) P1851 PIPES
85 mm KCl P9541 Potassium chloride
0.5% NP-40 NP40 Tergitol®
1 mm PMSF (phenylmethylsulfonyl fluoride) P7626 Phenylmethanesulfonyl fluoride
Nuclear lysis buffer      
50 mm Tris-HCl (pH 8) T5941 Trizma® hydrochloride
10 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
0.8 % SDS L4390 Sodium dodecyl sulfate
1 mm PMSF (phenylmethylsulfonyl fluoride) P7626 Phenylmethanesulfonyl fluoride
Dilution buffer      
10 mm Tris-HCl (pH 8.0) T5941 Trizma® hydrochloride
0.5 mm EGTA E3889 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
1% Triton X-100 T8787 Triton® X-100
140 mm NaCl S3014 Sodium chloride
1 mm PMSF P7626 Phenylmethanesulfonyl fluoride
RIPA buffer      
10 mm Tris-HCl (pH 8.0) T5941 Trizma® hydrochloride
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
0.5 mm EGTA E3889 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
1% Triton X-100 T8787 Triton® X-100
0.1% sodium deoxycholate D6750 Sodium deoxycholate
0.1% SDS L4390 Sodium dodecyl sulfate
140 mm NaCl S3014 Sodium chloride
1 mm PMSF P7626 Phenylmethanesulfonyl fluoride
LiCl buffer      
0.25 m LiCl L9650 Lithium chloride
0.5% NP-40 NP40 Tergitol®
0.5% sodium deoxycholate D6750 Sodium deoxycholate
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
10 mm Tris-HCl (pH 8.0) T5941 Trizma® hydrochloride
TE      
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
10 mm Tris-HCl (pH 8.0) T5941 Trizma® hydrochloride
Gel loading solution      
0.25% bromophenol blue B0126 Bromophenol Blue
0.25% xylene cyanol X4126 Xylene Cyanol FF
30% glycerol G5516 Glycerol
Ligase buffer      
12.5 mm MgCl2 M8266 Magnesium chloride
25 mm dithiothreitol (DTT) D9779 DL-Dithiothreitol
1.25 mm ATP A6559 Adenosine 5'-triphosphate disodium salt solution
50 mm Tris-HCl (pH 7.6) T5941 Trizma® hydrochloride
Hybridization buffer      
7% SDS L4390 Sodium dodecyl sulfate
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
1% bovine serum albumin B4287 Albumin from bovine serum
Wash buffer      
5% SDS L4390 Sodium dodecyl sulfate
1 mm EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
0.5% BSA B4287 Albumin from bovine serum
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