Protocols and Applications Guide (US Letter Size) - Promega

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||||||||||| 11Protein Purification and Analysis Antibody TF Experimental A/G TF TF TF Transcription Factor TF TF A/G TF Control TF TF TF TF Crosslink with Formaldehyde Lysis of Cytoplasm Nuclei Lysis in 1% SDS Sonicate Split Sample Incubate +/- Antibody O/N at 4°C Incubate with Protein A/G Agarose 2 hours at 4°C Wash Elute with TE +1% SDS Proteinase K, Reverse Crosslinks—O/N at 65°C Purify DNA Figure 11.10. Overview of chromatin immunoprecipitation using antibodies. Cells are grown using the appropriate conditions to form an interaction between the transcription factor (TF) of interest and DNA. To preserve the DNA:protein association during cell lysis, formaldehyde is added, resulting in crosslinks between the DNA and protein. A whole-cell extract is prepared, and the crosslinked chromatin is sheared by sonication to reduce the average DNA fragment size. A polyclonal or monoclonal antibody that recognizes the target protein is added, then incubated overnight. Protein A or Protein G agarose beads are added to capture the complex, then washed. The antibody must specifically and tightly bind its target protein under the wash conditions used. Finally, reversal of the formaldehyde crosslinking by heating permits the recovery and quantitative analysis of the immunoprecipitated DNA. One method of protein identification uses enzymatic digestion followed by mass spectrometry analysis. In this procedure, complex protein mixtures such as cell extracts are resolved by gel electrophoresis, and the band or spot of interest is excised from the gel and digested with trypsin. Trypsin is a serine protease that specifically cleaves at the carboxylic side of lysine and arginine residues. The distribution of Lys and Arg residues in proteins is such that trypsin digestion yields peptides of molecular weights that can be analyzed by mass spectrometry. The pattern of peptides obtained is used to identify the protein. Database searches can then be performed, using the mass of the peptides to identify the protein(s) resolved on the gel (Mann et al. 2001). The stringent specificity of trypsin is essential for protein identification. Native trypsin is subject to autolysis, generating pseudotrypsin, which exhibits a broadened specificity, including a chymotrypsin-like activity (Keil-Dlouha et al. 1971). Such autolysis products would result in additional peptide fragments that could interfere with database analysis of the mass of fragments detected by mass spectrometry. Protocols & Applications Guide www.promega.com rev. 3/09 6641MA Trypsin Gold, Mass Spectrometry Grade (Cat.# V5280), provides maximum specificity. Lysine residues in the porcine trypsin are modified by reductive methylation, yielding a highly active and stable molecule that is extremely resistant to autolytic digestion (Rice et al. 1977). The specificity of the purified trypsin is further improved by TPCK treatment, which inactivates chymotrypsin. The treated trypsin is then purified by affinity chromatography and lyophilized to yield Trypsin Gold, Mass Spectrometry Grade. More information and a detailed protocol are available in Technical Bulletin TB309 (www.promega.com /tbs/tb309/tb309.html). A. In-Gel Trypsin Digestion of Proteins Numerous protocols for in-gel protein digestion have been described (Flannery et al. 1989; Shevchenko et al. 1996; Rosenfeld et al. 1992). The following procedure has been used successfully by Promega scientists. Materials Required: (see Composition of Solutions section) • Trypsin Gold, Mass Spectrometry Grade (Cat.# V5280) and protocol PROTOCOLS & APPLICATIONS GUIDE 11-21
||||||||||| 11Protein Purification and Analysis HaloTag ® Vector Transfection HT TF HaloLink HaloLink Covalent Capture of Chromatin Complexes Using HaloTag ® Technology Experimental Sample HT HT TF TF TF HT TF HT HaloCHIP Blocking Ligand HaloLink HaloLink Control Sample TF HT Sample DNA Background DNA Analyze Analyze Expression of HaloTag ® Fusion Protein Formaldehyde Cross-linking Lysis, Sonication Split Sample Add HaloCHIP Blocking Ligand to the control sample to prevent binding to HaloLink Resin Capture on HaloLink Resin Wash HaloLink Resin Covalent capture allows for highly stringent washes to remove non-specific proteins and DNA Release of DNA by reversal of cross-links Figure 11.11. Capture of DNA:protein interactions using the HaloTag® technology. The protein-coding sequence of a transcription factor (TF) is cloned into a HaloTag® (HT) mammalian expression vector. This recombinant vector is transfected into mammalian cells, and the cells are grown under the appropriate conditions to allow formation of DNA:protein interactions. To preserve the DNA:protein association, formaldehyde is added, resulting in crosslinks between DNA and protein. A whole-cell extract is prepared, and the crosslinked chromatin is sheared by sonication to reduce the average DNA fragment size. The complex is then immobilized by adding the HaloLink Resin, followed by a short incubation. Reversal of the formaldehyde crosslinking by heating permits the recovery and quantitative analysis of immunoprecipitated DNA. • SimplyBlue SafeStain (Invitrogen Cat.# LC6060) • trifluoroacetic acid (TFA) • acetonitrile (ACN) • 200mM NH4HCO3 buffer (pH 7.8) • NANOpure® water • ZipTip® scx pipette tips (Millipore Cat.# ZTSCXS096) • α-cyano-4-hydroxycinnamic acid (CHCA) • MALDI target • ZipTip® C18 pipette tips (Millipore Cat.# ZTC18S096) Protocols & Applications Guide www.promega.com rev. 3/09 1. Separate protein samples by electrophoresis on an SDS-Tris-Glycine gel. 1–1.5 days Note: Other gel systems and staining reagents can be used for in-gel digestions but should be tested to ensure compatibility with the protein of interest and detection system being used. PROTOCOLS & APPLICATIONS GUIDE 11-22
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification is co
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification React
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Additional Res
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|| 2RNA Interference 2. Combine sep
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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||| 3Apoptosis Qi, H. et al. (2003)
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|||| 4Cell Viability CONTENTS I. In
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|||| 4Cell Viability E. Homogeneous
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||||| 5Protein Expression I. Introd
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||||||| 7Cell Signaling CONTENTS I.
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||||||| 7Cell Signaling SMALL GTP-B
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||||||| 7Cell Signaling HO S N N S
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||||||| 7Cell Signaling PN083 Intro
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|||||||| 8Bioluminescence Reporters
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Table 8.1. pGL4 Luciferase Reporter
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||||||||| 9DNA Purification I. Intr
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