Product Includes | Volume (with Count) | ||||
---|---|---|---|---|---|
PTMScan® IAP Buffer (10X) 9993 | 10 x 600 µl | ||||
PTMScan® Mono-Methyl Lysine Motif (mme-K) IAP Beads | 10 x 80 µl |
Product Information
Reagents Not Included:
NOTE: Prepare solutions for cell lysis (Section I), C18 column purification (Section II), and peptide binding and washing during IAP enrichment (Section III) with reverse osmosis deionized (RODI) or equivalent grade water. Prepare solutions using HPLC grade water (Burdick and Jackson water) for the peptide elution (Section III) and the peptide concentration steps (Section IV).
Stock Solutions:
NOTE: Prepare solutions with RODI or equivalent grade water.
NOTE: The urea lysis buffer should be prepared fresh prior to each experiment. Do not include protease inhibitors.
NOTE: Dissolving urea is an endothermic reaction. Urea lysis buffer preparation can be facilitated by placing a stir bar in the beaker and by using a warm (not hot) water bath on a stir plate. 9 M urea is used so that upon lysis, the final concentration is approximately 8 M. The urea lysis buffer should be used at room temperature. Placing the urea lysis buffer on ice will cause the urea to precipitate out of solution.
NOTE: If desired, the PTMScan® protocol may be interrupted at this stage. The harvested cells can be frozen and stored at -80°C for several weeks.
NOTE: Centrifugation is performed at room temperature to prevent urea from precipitating out of solution.
NOTE: Lysate sonication fragments DNA and reduces sample viscosity. Ensure that the sonicator tip is submerged in the lysate. If the sonicator tip is not submerged properly, it may induce foaming and degradation of your sample.
NOTE: DO NOT place urea lysis buffer or culture dishes on ice during harvesting. Harvest cells using urea lysis buffer at room temperature. During lysis, the buffer becomes viscous due to DNA released from the cells.
NOTE: If desired, the PTMScan® protocol may be interrupted at this stage. The cell lysate can be frozen and stored at -80°C for several weeks.
NOTE: Lysate sonication fragments DNA and reduces sample viscosity. Ensure that the sonicator tip is submerged in the lysate. If the sonicator tip is not submerged properly, it may induce foaming and degradation of your sample.
* To view an updated protease digestion reference table, please visit cellsignal.com/learn-and-support/protocols/ptmscan-motif-antibody-kits.
NOTE: Alternative proteases such as GluC, chymotrypsin, and others can be used in addition to the protease digests outlined in the reference table to expand the coverage of modified peptides from each Motif Antibody. When considering the use of additional protease digests, it should be compatible with the respective Motif Antibody by not cleaving residues within the designated sequence motif. Alternate protease digests that generate larger proteolytic peptides may not be ideal if the resulting peptides do not ionize well in the mass spectrometer.
NOTE: Purification of peptides is performed at room temperature on C18 reversed-phase columns from PTMScan® Peptide Purification Kit (Cell Signaling Technology, #35741).
NOTE: C18 purification uses reversed-phase (hydrophobic) solid-phase extraction. Peptides and lipids bind to the chromatographic material. Large molecules such as DNA, RNA, and most proteins, as well as hydrophilic molecules such as many small metabolites, are separated from peptides using this technique. Peptides are eluted from the column with 40% acetonitrile (ACN) and separated from lipids and proteins, which elute at approximately 60% ACN and above.
NOTE: About 20 mg of protease-digested peptides can be purified from one C18 column. Purify peptides immediately after proteolytic digestion.
NOTE: Prepare solutions with RODI or equivalent grade water. Use Trifluoroacetic acid (Thermo Fisher Scientific, 28904) and Pierce Acetonitrile (ACN), LC-MS Grade (Thermo Scientific, 51101) when preparing solutions. All percentage specifications for solutions are vol/vol.
NOTE: Organic solvents are volatile. Tubes containing small volumes of these solutions should be prepared immediately before use and should be kept capped as much as possible because the organic components evaporate quickly.
NOTE: Before loading the peptides from the digested sample on the column, they must be acidified with TFA for efficient peptide binding. The acidification step helps remove fatty acids from the digested peptide mixture.
NOTE: Application of all solutions should be performed by gravity flow.
NOTE: Each time solution is applied to the column, air bubbles form in the junction where the 10 cc syringe meets the narrow inlet of the column. These must be removed with a gel-loading tip placed on a P200 micropipettor, otherwise the solution will not flow through the column efficiently. Always check for appropriate flow.
NOTE: In rare cases, if the flow rates decrease dramatically upon (or after) loading of sample, the purification procedure can be accelerated by gently applying pressure to the column using the 10 cc plunger after cleaning it with organic solvent. Again, make sure to remove air bubbles from the narrow inlet of the column before doing so. Do not apply vacuum.
NOTE: Due to the relatively large sample peptide amounts and volumes at this stage of the protocol, the lyophilization should be performed in a standard lyophilization apparatus rather than a vacuum centrifuge (Speed-Vac) apparatus.
NOTE: The lysate digest may have a much higher volume than the 10 cc reservoir will hold (up to 50-60 mL from adherent cells), and therefore, the peptides must be applied in several fractions. If available, a 60 cc syringe may be used in place of a 10 cc syringe to allow all sample to be loaded into the syringe at once.
NOTE: Lyophilized, digested peptides are stable at -80°C for several months (seal the closed tube with parafilm for storage). The PTMScan® procedure can be interrupted before or after lyophilization. Once the lyophilized peptide is dissolved in IAP buffer (see next section), continue to the end of the procedure.
NOTE: Prepare solutions with RODI or equivalent grade water. Trifluoroacetic acid should be of the highest grade. All percentage specifications for solutions are vol/vol.
NOTE: After dissolving the peptide, check the pH of the peptide solution by spotting a small volume on pH indicator paper. The pH should be close to neutral, or no lower than 6.0. In the rare case that the pH is more acidic (due to insufficient removal of TFA from the peptide under sub-optimal conditions of lyophilization), titrate the peptide solution with 1 M Tris base solution that has not been adjusted for pH. Usually, 5-10 μL is sufficient to neutralize the solution.
NOTE: Some Phenol Red pH indicator may remain (it co-elutes during the C18 purification of peptides) and color the peptide solution yellow. This coloration has no effect on the immunoaffinity purification step.
NOTE: Perform all subsequent wash steps at 2-4°C. For all the washes except the final wash, avoid removing the last few microliters, since this may cause inadvertent removal of the beads.
NOTE: All steps from this point forward should be performed with solutions prepared with Water, LC-MS Grade (Burdick and Jackson) (Cell Signaling Technology #27732).
NOTE: After the last wash step, remove supernatant with a P1000 micropipettor as before, then centrifuge for 5 sec at 2,000 x g to remove fluid from the tube walls, and carefully remove all remaining supernatant with a gel loading tip attached to a P200 micropipettor.
NOTE: In this step, the post-translationally modified peptides of interest will be in the eluent.
NOTE: We recognize there are many other routine methods for concentrating peptides using commercial products such as C18 tips (see below) that have been optimized for peptide desalting/ concentration. Regardless of the particular method, we recommend that the method of choice be optimized for recovery and be amenable for peptide loading capacities of at least 10 µg.
C18 tips: Pierce C18 Spin Tips (Thermo Fisher Scientific, 84850)
NOTE: Prepare solutions with Water, LC-MS Grade (Burdick and Jackson) (Cell Signaling Technology #27732). Organic solvents (trifluoroacetic acid, acetonitrile) should be of the highest grade. Pierce Trifluoroacetic Acid (TFA), Sequencing grade (Thermo Fisher Scientific, 28904) and Pierce Acetonitrile (ACN), LC-MS Grade (Thermo Scientific, 51101) are recommended.
NOTE: Organic solvents are volatile. Tubes containing small volumes of these solutions should be prepared immediately before use and should be kept capped as much as possible because the organic components evaporate quickly.
Protocol Id: 2725
Methylation of lysine residues is a common regulatory post-translational modification (PTM) that results in the mono-, di-, or tri-methylation of lysine at ε-amine groups by protein lysine methyltransferases (PKMTs). Two PKMT groups are recognized based on structure and catalytic mechanism: class I methyltransferases or seven β strand enzymes, and SET domain-containing class V methyltransferases. Both use the methyl donor S-adenosyl-L-methionine to methylate histone and non-histone proteins. Class I methyltransferases methylate amino acids, DNA, and RNA (1,2). Six methyl-lysine-interacting protein families are distinguished based on binding domains: MBT, PHD finger, Tudor, PWWP, WD40 repeat, and chromodomains. Many of these display differential binding preferences based on lysine methylation state (3). KDM1 subfamily lysine demethylases catalyze demethylation of mono- and di-methyl lysines, while 2-oxoglutarate-dependent JmjC (KDM2-7) subfamily enzymes also modify tri-methyl lysine residues (4).
Most PKMT substrates are histone proteins and transcription factors, emphasizing the importance of lysine methylation in regulating chromatin structure and gene expression. Lys9 of histone H3 is mono- or di-methylated by G9A/GLP and tri-methylated by SETDB1 to activate transcription. JHDM3A-mediated demethylation of the same residue creates mono-methyl Lys9 and inhibits gene transcription (5). Tumor suppressor p53 is regulated by methylation of at least four sites. p53-mediated transcription is repressed following mono-methylation of p53 at Lys370 by SMYD2. Mono-methylation at Lys382 by SET8 suppresses p53 transcriptional activity, while SET7/9 mono-methylation at Lys372 inhibits SMYD2 methylation at Lys370 and stabilizes the p53 protein (1,6). Overexpression of PKMTs is associated with multiple forms of human cancer, which has generated tremendous interest in targeting protein lysine methyltransferases in drug discovery research.
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