Product Includes | Volume (with Count) | ||||
---|---|---|---|---|---|
PTMScan® Anti-Biotin (A7C2A) Immunoaffinity Beads | 10 x 80 µl | ||||
PTMScan® IAP Buffer (10X) 9993 | 10 x 600 µl |
Product Information
Reagents Not Included:
Reagents Specifically for APEX Biotin Labeled Samples:
NOTE: Prepare solutions for cell lysis (Section I), C18 column purification (Section II), and 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 concentration steps (Section IV and V).
Stock Solutions:
NOTE: Prepare solutions with RODI or equivalent grade water.
NOTE: For APEX biotinylated samples, add 10 mM sodium ascorbate, 5 mM Trolox, 10 mM sodium azide in Urea Lysis buffer. Make 1 M sodium ascorbate in water and 500 mM Trolox in DMSO immediately before making solutions. Do not store the stock. Make 1M sodium azide in water. Aliquots can be stored at -20 °C or below for several months.
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: For APEX biotinylated cells, wash cells with 25 ml of quenching solution (1xPBS with 10 mM sodium ascorbate, 5 mM Trolox, 10 mM sodium azide) three times before 1x PBS wash.
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: For APEX biotinylated cells, wash cells with 25 ml of quenching solution (1x PBS with 10 mM sodium ascorbate, 5mM Trolox, 10mM sodium azide) three times before 1x PBS wash.
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.
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 protein, 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 Peptide Purification column. Purify peptides immediately after proteolytic digestion.
NOTE: Prepare solutions with RODI or equivalent grade water. Use Trifluoroacetic acid (TFA), Reagent grade (American Bioanalytical, AB02010) and Acetonitrile (ACN), LC-MS Grade (Cell Signaling Technology, 95031) 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 P-200 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: The lyophilization should be performed in a standard lyophilization apparatus. DO NOT USE a SPEED-VAC apparatus at this stage of the protocol.
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 step), 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. 5-10 µl is usually sufficient to neutralize the solution.
NOTE: In the following steps, it is critical to centrifuge the beads at the recommended g-force for quantitative recovery and to avoid damage to the bead matrix.
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 Burdick and Jackson or other HPLC grade water.
NOTE: After the last wash step, remove supernatant with a P-1000 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 P-200 micropipettor.
NOTE: In this step, the biotinylated peptides will be in the eluent.
NOTE: We recognize there are many 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: Thermo Scientific, part number 84850
NOTE: Prepare solutions with Burdick and Jackson water or other HPLC grade water. Organic solvents (trifluoroacetic acid, acetonitrile) should be of the highest grade. Pierce™ Trifluoroacetic Acid (TFA), Sequencing grade (Thermo Scientific, 28903) 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: 2726
Biotinylation of proteins and peptides has been used for many years and has become a common way to biochemically tag targets of interest (1). One advantage is the very strong binding interaction with avidin that can be used to purify biotin-tagged proteins (2,3). It can also be used as a method for conjugation of a variety of tags such as dyes, beads and other solid substrates for a variety of assays and tools (4,5).
New assays have been developed that improve elucidation of protein subcellular context, localization, and protein-protein interactions. These new cellular assays utilize biotin ligase or engineered ascorbate peroxidase (APEX) fusion proteins that are able to biotinylate nearby protein targets within specific cellular compartments. This type of biotin proximity assay can provide improved information about subcellular localization, multi-subunit components, and recruitment of unknown targets. Using CST's in house developed anti-biotin rabbit monoclonal antibody; we have developed a robust procedure for immunoaffinity enrichment followed by mass spectrometry analysis using CST’s proprietary PTMScan(R) protocols. This allows for identification of biotinylated targets and investigation of new protein interactions. This assay provides rich data for these types of biotin proximity assays such as the APEX (6,7) engineered cell system and other related methods.
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