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Proteomics

CelLytic PN


Description:

This kit is for the rapid isolation of nuclei and extraction of functional nuclear proteins from plant leaves. Nuclei or nuclear proteins can be extracted from a few grams to hundreds of grams of fresh and frozen leaves.

The nuclear protein extract is suitable for the detection of DNA-protein interactions using gel-shift assay, DNase-I footprinting analysis as well as Western blot assay and similar techniques. The isolated nuclei can also be used as a source for chromatin, genomic DNA, RNA, etc. The kit provides a detailed protocol for nuclei isolation and protein extraction from four plant models: tobacco, tomato, spinach, and Arabidopsis.

 
Kit Components:
  • Nuclei Isolation Buffer 4x (NIB)
  • Percoll
  • Sucrose 2.3 M
  • TRITON®
  • Extraction Buffer
  • Nuclei PURE Storage Buffer
  • Filter Mesh 100


Figure 1. Detection of RNA polymerase II in Tomato Nuclear versus Cytoplasmic extracts, prepared with CelLytic-PN Kit. The Extracts were run on SDS-PAGE and blot-hybridized to anti-RNA Polymerase II antibody.

Compatibility with Gel Shift Assay

Figure 2. Gel Shift analysis of protein-DNA complex formed between CREB DNA binding site and CREB protein in Tobacco Nuclear extracts. Protein extracts were prepared with the CelLytic-PN kit from nuclei isolated at three levels of purity (high, semi-pure, crude). A double stranded 32P labeled CREB oligonucleotide probe was incubated with 1 µg of cytoplasmic or nuclear extract. Binding reactions with the nuclear extracts were performed in the absence [-] of competitor oligonucleotide (lanes 1-5) or in the presence of x100 or x500 fold excess of unlabeled CREB binding motif oligonucleotide (specific competitor [SP], lane 6 and lane 7, respectively) or in the presence of x100 fold excess of unlabeled oligonucleotide (non specific competitor, [NS] lane 8).
Lane 1: Free probe without extract.
Lane 2: Cytoplasmic fraction present in the supernatant of "High" level of purity.
Lane 3: Nuclear proteins isolated by "High" level of purity.
Lane 4: Nuclear proteins isolated by "Semi-pure" level of purity.
Lanes 5-8: Nuclear proteins isolated by "Crude" level of purity.
Binding reactions were run on a non-denaturing 6% polyacrylamide gel, dried and imaged on film. The arrows indicate the CREB-DNA complex and the free probe.

Ordering Information

Product Product Name Package Size Technical Bulletin
CELLYT-PN-1 CelLytic PN Extraction Kit 1 Kit X