Connect the elution tip to a vacuum via a piece of tubing and turn the vacuum on. Use the small end of the elution tip to scrape the cellulose off a spot of interest; the cellulose will be sucked up against the filter barrier in the tip as it is scraped from the plate. When the spot is completely removed from the plate, ease the tubing off the wide end of the elution tip, keeping the small end upright. The same spot can be vacuumed from multiple plates into one elution tip.
When this happens, use a razor blade to trim a thin sliver of plastic from the small end of the tip to recreate the sharp edge. Place the elution tip into a 1. The elution tip now becomes a little column. The elution buffer used here should be pH 1. If pH 1. Remove the elution tip from each of the microcentrifuge tubes, being careful to leave all the eluate in the tube some may cling to the sides of the tip as drops, which should be removed and added back to the contents of the tube.
Save the elution tip. If eluting more than one spot, keep track of which tip was used for which peptide. Clarify the eluate s by microcentrifuging 5 min at full speed a small cellulose pellet will be visible after centrifugation; its size will depend on how snugly the sintered disk fits into the elution tip. Transfer the supernatant to a fresh microcentrifuge tube. It is very important to remove all traces of cellulose at this point, as contamination of the phosphopeptide with cellulose can ruin further analyses.
Given the pain and frustration involved in their manufacture, a good elution tip should be saved and reused. Dry and then count the tips on a scintillation counter before Protein reusing them. Lyophilize the eluates in a SpeedVac, then count them by Cerenkov counting. The counts here should be slightly higher than those of the liquid eluate. The number of cpm in this final sample of eluted peptide will often determine how it can be analyzed further.
During each cycle of Edman degradation, the most amino-terminal amino acid residue is released from the peptide, and a sample from the reaction mixture is taken after each cycle. Phosphoserine or phosphothreonine is released as a derivative of serine or threonine and free phosphate; in contrast, phosphotyrosine is released as the anilinothiazolinone derivative of phosphotyrosine.
Free phosphate and the PTH derivative of phosphotyrosine can be separated from the peptide by electrophoresis on a TLC plate. This approach indicates at which cycle the radioactivity and thus the phosphorylated amino acid is released from the peptide. Decide the number of cycles to be run based on the list of candidate peptides. The number of cycles is designated as X. This sample will be lyophilized with the other cycle fractions at a later point. Perform the Edman reactions 4.
Count Phosphopeptide the sample at this point: Mapping and Identification of a. Microcentrifuge 1 min at full speed to separate the two phases. The pyridine will partition into the upper organic phase. Carefully remove the upper organic phase using a plastic transfer pipet. Freeze the aqueous phase on dry ice and lyophilize in a SpeedVac evaporator.
Lyophilize the sample in a SpeedVac evaporator. Count the sample by Cerenkov counting. There should be the same number of cpm as at the beginning of the cycle i. Repeat steps 5 to Repeat steps 4 to Continue repeating steps 4 to 12 until the desired number of cycles have been run. Analyze the Edman products Lyophilize all samples to dryness in a SpeedVac evaporator. Count all final samples by Cerenkov counting, Microcentrifuge 2 min at maximum speed to bring down any insoluble material.
Alternatively, if the sample volumes removed after each cycle are small enough, skip steps 17 and 18 and load the samples directly onto the TLC plate. Spot all samples from the analysis of a given phosphopeptide at least 1 cm apart on a line of origins running vertically through the center of the TLC plate Fig. A Location of the sample and standard origins. To mark a TLC plate, the plate is placed on top of a life-size template on top of a light box and the origins are marked on the cellulose side using a very blunt extra-soft pencil.
B Dimensions of the blotter and the location of the slot that fits over multiple sample and marker origins. The blotter is soaked in electrophoresis buffer, blotted with a sheet of Whatman paper to remove most of the buffer, and placed on top of the TLC plate so that the origins are in the middle of the slot. Wet the plate as described in Figure Prepare the HTLE apparatus and electrophorese the samples for 25 min at 1.
After incubation with a protease or chemical, the peptide is analyzed by separation in two dimensions on a TLC plate. A change in mobility upon treatment with a particular reagent indicates that the peptide was susceptible to cleavage, and consequently that the amino acid or amino acid sequence that confers susceptibility to cleavage by this reagent must be present in the peptide.
Materials Phosphopeptide Eluted phosphopeptide see Support Protocol 1 Mapping and Enzyme to be used for digestion and appropriate buffer see Table Incubate all tube s in a water bath at the appropriate temperature for at least 1 hr.
Add another aliquot of enzyme and continue the incubation step for an additional hour. It is necessary to completely inactivate the enzyme prior to loading the sample on the plate when analyzing a mix of digested and undigested peptide, since the undigested sample may Analysis of be rapidly digested when the two samples are mixed.
Lyophilize the samples in a SpeedVac evaporator. Microcentrifuge at full speed to bring down insoluble material. Load half of the undigested sample on a single TLC plate. Load half of the digested sample on each of two TLC plates; on one of these load the other half of the corresponding undigested sample as a mix.
Perform electrophoresis and chromatography on the plate as described above in Basic Protocol 1, steps 24 to Based on the position where the particular phosphopeptide being analyzed ran in the original map, choose a pH and running time that will allow good separation of the peptide from its potential cleavage products but will ensure retention of the smaller cleavage products on the plate.
Optimize 32P labeling of the protein of interest. If the site of interest is seen only in stimulated cells, a time course of phosphorylation following stimulation may be helpful, as would determination of the optimal concentration of agonist. If an in vitro system is being employed, determine the optimum conditions time, and ratio of kinase and substrate concentrations for the kinase reaction. Include 1 mM cold ATP in the reactions to maximize the stoichiometry.
UNIT Calculate the number of cells or amount of substrate needed to isolate 10 pmol phosphorylated material. In intact cells, the stoichiometry may be even less. It cannot hurt to overestimate the amount of starting material required, as the losses taken during the isolation procedures will always exceed expectation. When calculating how to scale up the reactions, consider the following points. The radioactivity of these samples is only used for visualization purposes—i.
When isolating overexpressed protein from cells, labeling only 2 or 3 dishes of the 20 needed to generate enough material may be sufficient. To generate enough material for further analysis, perform an additional kinase reaction with unlabeled ATP only. Mapping and Identification of Phosphorylation Sites The efficiency of protein elution decreases as the amount of gel increases, so try to keep the number of lanes on the preparative gel s to a minimum. This will result in a cleaner sample, as the tryptic fragments of the carrier protein will be eliminated from the mix of fragments run on the TLC plate.
While it is important that the digestion go as far to completion as possible, it is probably not necessary to scale up the amount of trypsin used. Instead, consider pooling several like samples at the performic acid digestion step at the end of the 60 min incubation in order to give the protein the maximum time to dissolve.
Determine the number of TLC plates to be run based on the amount of total protein to be analyzed—total protein includes the amount of trypsin and the amount if any of carrier protein used as well as the amount of the protein of interest. Chromatography buffers Phosphochromatography buffer: ml n-butanol ml pyridine ml glacial acetic acid ml deionized water Store at room temperature Isobutyric acid buffer: ml isobutyric acid 38 ml n-butanol 96 ml pyridine 58 ml acetic acid ml deionized water Regular chromatography buffer: ml n-butanol ml pyridine ml glacial acetic acid ml deionized water Store all of the above buffers up to 6 months at room temperature.
Record the pH and the date on the bottle; if the pH is more than a tenth of a unit off, remake the solution. Do not adjust the pH. Store all buffers at room temperature. Store up to 1 year at room temperature. Freeze the aqueous phase and lyophilize in a SpeedVac evaporator.
Dissolve the sample in 0. Dissolve in 1 ml pH 1. PVP in mM acetic acid 0. All three of these techniques Phosphopeptide mapping is a very sensitive are easily accomplished in a laboratory that is technique that can help the investigator answer already set up for phosphopeptide mapping.
The first step in all three is the isolation of the For some, phosphopeptide mapping is a tool to phosphopeptide from the cellulose plate see find out whether a particular protein is phos- Support Protocol 1. This question can be tested by phosphopeptide mapping of a can be answered by simply running a phos- mutant protein lacking a phosphate acceptor at phopeptide map of the protein labeled in living the site in question.
Alternatively the guess can cells. Other investigators want to know whether be substantiated by synthesizing the tryptic the increase in phosphorylation seen when cells phosphopeptide and testing it for comigration are treated with a particular agent is restricted with the phosphopeptide isolated from the pep- to one or more specific sites or whether it is tide map. Finally, detailed Critical Parameters and analysis of phosphopeptides isolated from a Troubleshooting TLC plate can be used to identify the residues that are phosphorylated in a protein of interest.
Generating phosphopeptide maps Several different strategies may be followed Keep in mind that the sort of analyses pre- to identify the phosphorylation site represented sented throughout this unit will give informa- by a particular spot on a phosphopeptide map.
While these two techniques re- Carrier Protein. The authors prefer to use quire expensive instruments and expertise not RNase as carrier protein during TCA precipi- found in most laboratories, such analysis can tation, particularly when analyzing proteins la- often be arranged by collaboration.
However, beled in intact cells, because it degrades 32P-la- sometimes it is not possible to take advantage beled RNA species that may have copurified of these techniques, since they require 1 to 10 with the protein of interest.
The nucleotides pmole of material for analysis. For a kDa generated by the degradation of RNA do not protein one would need 0. In order to generate a stoichiometry of phosphorylation at the site of phosphopeptide map, the 32P-labeled protein interest. Use of an in vitro phosphorylation needs to be cleaved into smaller fragments that system that mimics the situation in intact cells can be separated by electrophoresis and chro- will simplify matters greatly.
Further consid- matography on TLC plates. To do this requires erations and strategies for preparation of sam- an enzyme or chemical agent that cleaves re- ples for these two techniques are discussed at producably and with a certain frequency. If not the end of this chapter in Support Protocol 2. In addition, large identity include phosphoamino acid analysis of fragments may contain multiple phosphoryla- the individual phosphopeptide UNIT This leads to maps that are less result of manual Edman degradation of a phos- informative and more difficult to analyze.
The phopeptide providing the cycle at which the authors routinely use trypsin and chymotryp- phosphate is released and thus the position of sin. Other reagents are available Table Follow- Analysis of of other specific amino acids in the peptide see ing digestion, repeated cycles of lyophilization Protein Phosphorylation The authors like to load bicarbonate.
The presence of salts in the sample at least cpm on a plate for a peptide map. The presence of any crys- sample. Remember that overloading can lead talline material indicates the presence of salts, to streaky maps.
If a preparative map from most likely ammonium bicarbonate that can be which a particular peptide will be isolated is removed by additional rounds of lyophiliza- being run, it may be best to run the entire sample tion. Theoretically, Controlling oxidation. The oxidation state of these amino the case in practice. Check the rate at which the acids affects the mobility of peptides during first drop spotted sinks into the cellulose; as chromatography, resulting in separation of oxi- more sample is spotted, this rate will decrease.
This complicates the in- If, while spotting, one gets to a point where the terpretation of the phosphopeptide map. To sample drop just sits on the origin and does not prevent this, the protein or peptides are oxidized spread into the cellulose, stop loading.
Incu- Peptide diffusion. Peptides diffuse during bation at higher temperatures may give rise to the electrophoresis and chromatography, and unwanted side reactions and should be avoided. To counteract this, the authors try to a membrane? In Basic Protocol 1, the 32P-la- keep the area on the TLC plate onto which the beled protein is isolated from a small slice of a sample is spotted as small as possible by spot- dried polyacrylamide gel by rehydrating and ting only a small amount at a time i.
In addition, the sample is concen- The protein is subsequently TCA precipitated, trated by wetting the TLC plates with electro- oxidized, and digested with trypsin. This is a phoresis buffer using a blotter with holes cut time-consuming and laborious procedure.
The alternative is to transfer the protein results in buffer moving from the blotter to- to a PVDF membrane; any unoccupied protein- wards the center of the hole.
This concentrates binding sites on the strips of membrane con- the sample on the origin. For this process to taining the protein of interest are blocked by work well, the origin has to be precisely in the incubation with PVP in acetic acid before center of the hole.
In addition, the buffer has to incubation with trypsin. Most peptides dis- move with similar speed from the entire cir- lodge from the membrane during the digestion. The sample will This protocol is much faster and less laborious, inevitably streak if the buffer takes a long time and does not require the use of additional carrier to wet the spot, or moves unevenly through the proteins that may lead to overloading of the spot.
TLC plate and to streaky maps. Obviously this Electrophoresis system. This system features water membrane. In addition, it is possible that certain cooling and an inflatable airbag that presses the peptides that are generated during protease di- TLC plate against the cooling plate.
Water cool- gestion retain a high affinity for the membrane ing prevents overheating during electrophore- and therefore fail to elute. If those peptides sis. The inflatable airbag presses excess buffer contain a phosphorylation site, this site will not from the TLC plate; this limits diffusion of the be represented on the peptide map. This can peptides and improves resolution. It is Buffers. Three different buffers are typically therefore advisable to first compare maps gen- used for electrophoresis: pH 1.
Less often used are pH 3. If these maps are identical, and if the electrophoresis buffer. To find out which buffer Mapping and protein transfers well from the gel to the mem- gives the best separation of peptides generated Identification of Phosphorylation brane, the Alternate Protocol should be the from a particular protein, all three buffers Sites protocol of choice.
If possible, the authors prefer Most peptides Phosphopeptide identification dissolve well at this pH. In addition, use of this After running several phosphopeptide buffer results less often in streaky maps. Sign up Log in. Web icon An illustration of a computer application window Wayback Machine Texts icon An illustration of an open book. Books Video icon An illustration of two cells of a film strip.
Video Audio icon An illustration of an audio speaker. Audio Software icon An illustration of a 3. Software Images icon An illustration of two photographs. This four-volume laboratory manual contains comprehensive state-of-the-art protocols essential for research in the life sciences. Techniques are presented in a friendly step-by-step fashion, providing useful tips and potential pitfalls.
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DNA Isolation 2. Electrophoresis 3. Isolation of total RNA 4. Restriction Endonucleases 5. Hybridization Techniques 7.
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