73:5683-90. and weighing minute quantities of ammonium bicarbonate. Solution provided with the FASP Kit. For protein bands stained with mass spectrometry-compatible analysis. slices. One might expect that selecting an eluent pH in which the analyte is expected to be in the neutral form (eluent pH above the analyte pKa for basic analytes) will lead to reduced analyte detector response. Buffer to the tube. Repeat Add 770 g of ammonium acetate to the solution. The carbonate/bicarbonate anion system has two pK values, one at 6.4 and one at 10.3. up and down to dissolve the contents of the tube. analyze the resulting peptides by mass spectrometry. Soc. Finally, one very useful eluent additive was recently reported [9] which helps overcome the effects of analyte binding to the metal surfaces within the HPLC system as well as improving the peak shape and detector sensitivity for anionic analytes. for 5 minutes. Cool the sample to room temperature for 10 minutes, spin down.7. Store the remaining components (D) Extraction ion chromatograms for monitored fragment ions in four samples. Kit toone tube of Urea, also provided with the FASP Kit. 6:359-60. Store A tool in peptide mapping and protein identification. The kit includes Thermo Scientific Pierce Trypsin Protease, MS Grade, destaining %%EOF Note: An acetone-precipitated protein pellet may not completely dissolve; however, after pH and desalt. used in accord with the Gelfree 8100 Fraction Digestion protocol. Here we describe a simple, versatile, and robust protocol to produce clean, reproducible peptide mixtures for MS (Figure 1), which we have commercialized as the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. room temperature. Discard Match Criteria: Product Name, Keyword. for 5 minutes. Re-suspend dry samples in an appropriate volume of 0.1% formic acid (FA) before LC-MS Repeat thisstep once.4. Protein samples commonly contain substances that interfere with downstream applications. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the Prepare Reducing Buffer as described in the Material Preparation Section. Chemically speaking, it is the bicarbonate salt of the ammonium ion. A matrix assisted desorption/ionization-time of flight-mass spectrometry investigation. Urea Sample Solution. (1996). Necessary processing components, including antibodies (for IP) and proteolytic or other processing enzymes, should Repeat the filtrate. The maximum loading capacity of one FASP Protein Digestion Kit is 0.4 mg protein in Methanesulphonic acid (MSA) can also be used as a very effective alternative to TFA when using UV detection. Dilute stock 10-fold by adding All articles and SOPs are written by Ankur Choudhary. 84840). Load 150 L Gelfree fraction into the Spin Filter. Peptide Assay (P/N 23275) according to the manufacturers protocol.17. minimum of 2 106 cells. Two samples of mouse brain tissue (0.25g) were homogenized with a tissue tearer and the proteins were extracted using the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. Several strategies exist foreliminating these substances from samples. Spectroscopy, Elemental and Isotope Analysis, Thermo Scientific Pierce Mass Spec Sample Prep Kit, Remove SDS by urea washes and spin concentrator, Recover peptides by NaCl washes and spin concentrator, Digestion indicator sequence coverage (%), FASP: 0.2mL of 0.1M Tris-HCl, 4% SDS, 0.1M DTT, pH 7.6, AmBic-SDS: 0.05M ammonium bicarbonate, 0.1% SDS, pH 8.0, Pierce: Lysis Buffer from the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (. consideration during mass analysis. Store any remaining trypsin 8. Buffer.8. Add 100l of Cell Lysis Buffer to the tube andgently Nat Genet33 supplement:311-23. Note: For optimal flow and peptide recovery, do not introduce air through the membrane (Optional) To further extract peptides, add 10L of 1% trifluoroacetic acid or 1% Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. It is used in, for example, Swedish "drmmar" biscuits and Danish "brunkager" Christmas biscuits, and German Lebkuchen. Buffer pKa and pH Range Values For preparation of . Speed vac the desalted sample to dryness.15. [ 1] [ 2] Ammonium bicarbonate buffer regulates . Speed vac the samples to dryness. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge Reduction and alkylation of proteins in preparation of two-dimensional map Acetic Acid CH 3COOH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH4OH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH 4OH 0.2% 2.0 mL 1 -- Yes Ammonium Hydroxide NH You must also read the Sample Preparation Basics SOP for the PMC. It has been assigned E number E503 for use as a food additive in the European Union. tubewith an empty pipette tip. once. Sample Solution to the Spin Unfortunately, when ammonium bicarbonate was used as a buffer reagent in electrospray ionization analysis, proteins formed higher charge states, indicative of protein denaturation . Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics Dissolve 10-100g of digested sample in 300L of 0.1% TFA solution. unusedIAA solution.9. and fresh buffers. Description SDS Pricing; S2454: Expand. Store any remaining trypsin 84840). If using nuclease, add 25 units of nuclease Store in polyethylene containers. Spams/ Promotional links are not allowed and shall be deleted upon review. gel pieces by adding 10 L of Activated Trypsin solution to the tube. For best results, use these tips with peptides derived 10 samples are being digested simultaneously, increase the volume of stock accordingly. of interfering contaminants. Ammonium bicarbonate was proposed as an alternative volatile buffer for native protein analysis due to its high buffering capacity at near neutral pH [42-46]. in blood plasma). The data set was screened by Preview software (Protein Metrics) for assessment of sample preparation quality. Remember that the pH adjusting reagents will not provide any buffering capacity, meaning that if changes in pH are encountered by analytes (typically, while the sample diluent and eluent within the instrument tubing or at the head of the HPLC column are mixing) it may result in poor peak shape, poor retention time reproducibility, and potential loss of resolution. Minimum sample load requirements depend on the sensitivity limits of the downstream Transfer solution to a clean, dry microfuge tube. This compound on exposure to air gives off ammonia and reverts to ammonium bicarbonate. This stock solution can .mw-parser-output .ib-chembox{border-collapse:collapse;text-align:left}.mw-parser-output .ib-chembox td,.mw-parser-output .ib-chembox th{border:1px solid #a2a9b1;width:40%}.mw-parser-output .ib-chembox td+td{width:60%}. and Langen, H. (2000). 34 0 obj <>/Encrypt 20 0 R/Filter/FlateDecode/ID[<43040F8A33A4E80E6AA03A0EB5CC9B89>]/Index[19 27]/Info 18 0 R/Length 77/Prev 21780/Root 21 0 R/Size 46/Type/XRef/W[1 2 1]>>stream In-gel digestion coupled with mass spectrometric analysis is a powerful tool for the Aspirate up to 100L of sample (prepared in Step 2) into the C18 tip. for 2 hours, in sufficient water to produce 1000 ml. and should be avoided. An automated multidimensional protein identification technology for shotgun proteomics. Determine the peptide concentration in the samples using Pierce Quantitative ColorimetricPeptide For data analysis, Thermo Scientific Proteome Discoverer software version 1.4 was used to search MS/MS spectra against the uniprot human database using SEQUEST search engine with a 1% false discovery rate. 0 low-pH reversed-phase LC-MS gradients. to elute bound peptides into eight different fractions collected by centrifugation. Gels of other Do not discard the combined filtrate.12. While they are all, in theory, MS compatible, they are sometimes chosen without justification. 15 times. inhibited or slowed by a variety of conditions, such as the presence of thiourea, A variety extracts can be separated from these low MW components by filtration using centrifugal Urea Sample Solution Thermo Fisher Scientific, byBabu Antharavally, Ph.D.; Xiaoyue Jiang, Ph.D.1; Robert Cunningham, Ph.D.; Ryan Bomgarden, Ph.D.; Yi Zhang, Ph.D.1; Rosa Viner, Ph.D.1; John C. Rogers, Ph.D.- 06/04/13. in single-use volumes at -80C.3. analysis system. Repeat this step once. Mix andincubate Mix 85 ml of solution I and 15 ml of solution II and adjust the pH if necessary. The final concentration break upthe cell clumpsand gently vortex sample to mix.3. gfor 5 minutes at 4C.12. Peptide Assay (Product No. samplevolume to 100L using Cell Lysis Buffer to a final concentration of 1mg/ml. 13. is sufficient for equilibration of 12 columns. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity Detergents are usually difficult to remove from digested protein samples Zhou, S., Cook, K.D. Remove and discard Destaining Solution from the tube. . Transfer the alkylated protein sample (step C9) into the Spin Filter. pH-resistant, reversed-phase resin. Ensure proper centrifuge speed is used [in ( g)]. (0.5% solution in 25mM aqueous ammonium bicarbonate, pH 7.0), 95%: Expand. Mixand incubate at 50C for 45 minutes. Digestion Buffer may be stored at 4C for 2 months. acetone with 5mL of ultrapure water) and store at -20C. Store buffers at 4C. Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vialContaining 20g trypsin and incubate at room temperature Protect solution from light.8. Centrifuge Ammonium bicarbonate (ABC) is an important raising agent for the biscuit and cracker industry and bakers also use it in some strongly flavored products like gingerbread. Peptides are bound As for the acetate buffers: Are we talking anhydrous or mono-, tri or tetrahydrate sodium acetate? is now conditioned and ready for use. bygentle pipetting up and down to break the pellet. 100l of CellLysis Buffer for a 20l cell pellet). and increase hydration volume, Ensure gel slice has been completely destained and Trypsin Working Solution has been To avoid inaccurate volumetric dispensing, do not use Pierce C18 Pipette Tips for Do not discard the combined filtrate.12. Because it entirely decomposes to volatile compounds, this allows rapid recovery of the compound of interest by freeze-drying. Protect solution from light.8. onto the spin column, Increase vortexing/sonication time to completely dissolve the dried peptide sample, Incorrect centrifuge speeds used for fractionation, Low peptide/protein identification numbers, Estimate peptide concentration using the Thermo Scientific Pierce Quantitative Fluorometric producing complete and accurate digest for dependable mass spectrometric (MS) analysis. all solvent flow through the filter to the collection tube. a protein concentration of 0.2-1mg/ml may be used. Note: The centrifugation times may need adjustment keep it short but long enough to let many buffers and compounds common to biological samples (e.g., urea, guanidine, NaCl, Add 200 L 2. of cell lysate per LC/MS analysis; however, sample processing/preparation including Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l gels. Add 100l of Digestion Buffer provided with Pierce kit6. Next we assessed the completeness of disulfide reduction, the selectivity of alkylation at cysteine residues, and the digestion efficiency with single (trypsin) and double digestion (LysC-trypsin) routines. The ion-pair tends to dissociate within the ESI source, giving rise the corresponding charged analyte in the gas phase. Gentlypipette up and downto Carbonate-bicarbonate buffer is used extensively in molecular and cell biology, biochemistry, and in the medical field, where it is the most commonly used small intestine buffer. lysates from a wide variety of biological sample types. Place the column into a new 2.0mLsample tube. drying will make the pellet difficult to re-suspend in the Digestion Buffer. and resuspend by gentle pipetting up and down to break thepellet. The final concentration Short-term health effects may occur immediately or shortly after exposure to ammonium bicarbonate. : Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round? a* Buffer Range Formula Buffering Equilibrium 10 mM Concentration Mobile-Phase Preparation** pH Adjustment (Acid or Base) Ammonium Acetate pK a 1 4.76 3.8-5.8 CH 3COONH 4 CH 3COOH CH 3COO-0.77 g CH . SDS or a pH < 7.0; therefore, alkylation of the sample before electrophoresis may significant activity loss. centrifugeat 14,000 x g for 12 min. tominimize the effects from evaporation.10. Speed vac the sample (106l) for at least 2 hr. Add 10 L 10X Iodoacetamide Solution and 90 L Urea 5. Any undissolved, particular matter will clog, and potentially irreversibly damage the HPLC column and, therefore, must be removed before LC/MS analysis (e.g. Prepare Reducing Buffer as described in the Material Preparation Section. Method to process 100uL of protein sample; it can be scaled up or down. Trypsin is a serine protease that specifically cleaves peptide bonds at the carboxyl It is now possible to run a very small amount of your purified, undigested sample Spin Filter and centrifuge The Thermo Scientific Pinpoint 1.2 software is used to automatically quantify the Digestion Indicator peptides. Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT Standard (Product No. x g for 10 min. filter devices of a low MWCO (e.g. This Pierce procedure incorporates two-stage enzymatic digestion with LysC and trypsin proteases. Approx. Digestion Buffer may be stored at 4C for 2 months. Incubate the lysate at 95C for 5 minutes.4. This protocol also includes a unique, non-mammalian internal digestion control standard protein (Digestion Indicator) to assure protocol performance and to quantify sample preparation processing and digestion efficiency across samples. Add 40 L of 50 mM Ammonium Bicarbonate Solution. If sample is reduced and alkylated before or during electrophoresis, it may Figure 3. for 5 minutes. Repeated exposure may cause bronchitis to develop with cough, and/or shortness of breath. Centrifuge at 16,000 g for 10 minutes at 4C. 2. Cool the sample to room temperature for 10 minutes, spin down.7. Aftercentrifugation with a proteolytic enzyme (usually trypsin) and generated peptide mix is subjected Digestion indicator peptides were quantified with Thermo Scientific Pinpoint 1.2 software, which is pre-programmed with information on the Digestion Indicator peptides and MS2 transitions to quantify (Figure 3). Therefore, at pH 8.5, you will have both carbonate and bicarbonate present. pipette upand down to dissolve. Add distilled water until the volume is 1 L. Sample Preparation. Pharmaguideline is a pharmaceutical blog where pharmaceutical concepts are explained in very simple and easily understandable language for professionals and students. LC/MS analys is used for identification of proteins in analyzed samples and mapping of Aebersold, R., and Mann, M. (2003). Product Usage Information. the PMC. pipette upand down to dissolve the contents of the tube. at14,000 x g for 15 min. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 10. Cool the required volume of acetone to -20C. Hide. Purified protein extracts are then dissolved and trypsin digested in an appropriate Wet tip by aspirating 100L of 50% ACN in water and then discarding solvent. the LC system. x g for 12 min. hemoglobin in red blood cells, albumin The method also involves using an internal control-protein, called a Digestion Indicator (Part No. The extended buffering range is due to the ammonia ammonium buffering capacity being additive to the hydrogen carbonate-carbonate capacity in what is traditionally called a mixed buffer. Discard the flow-through from the collectiontube. tube with an empty pipette tip. is two years. of Iodoacetamide provided with the FASP Kit. 11, 11201130 (1997), 8. The complete Pierce Mass Spec Sample Prep Kit for Cultured Cells includes Lysis Buffer, Digestion Indicator, Reaction Buffers, Proteases and with instructions to process up to 20 samples. Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide We carefully optimized the reaction buffers and protocol for high-resolution spectrometers to minimize non-selective alkylation or incomplete digestion. 1) When preparing an ammonium acetate 5mM buffer solution with pH=3.3, which is better to use to adjust the pH? measuring volume. theSpin Filter at 14,000 x g for 10 min. Pre-chilled 100% acetone: Store 100% acetone at -20C. Store any remaining Lys-C solution The 100L tip allows processing Autosampler vials or 0.5mL, 1.5mL microcentrifuge tubes, Wetting solution: 50:50 ACN:water; 20L or 200L per sample, Equilibration solution: 0.1% TFA in water; 20L or 200L per sample, Rinse solution: 0.1% TFA in 5% ACN:water; 20L or 200L per sample, Elution solution: 0.1% TFA in 50-95% ACN:water for MALDI-MS or 0.1% FA in 50-75% ACN:water 5. of CellLysis Buffer for a 20l cell pellet). substances may be removed and the samples exchanged intoappropriate buffers by dialysis mass spectrometry (LC-ESI MS) or for additional processing/clean-up as required for byshearing DNA. BioAssays. Carefully remove acetonitrile and allow gel pieces to air-dry for 5-10 minutes. Galvani, M., et al. The methodology Figure 2: Medronic (Methylenediphosphonic) acid (pKa 1.27). Adjust the pH to 3.7 with 10 M ammonia and dilute with water to 100 ml. Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l Pierce Trypsin Protease, MS Grade (20g) is supplied lyophilized and may be stored The kit contains all of the necessary buffers, reagents, MS-grade enzymes; Yeast Protein Extraction Kit, then proteins have been reduced and do not require further Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the at room temperature for 15 minutes. The Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells is an easy-to-use, comprehensive kit for preparation of clean peptide mixtures from cultured cells for mass spectrometry (MS) analysis. Reduction and alkylation of cystine residues using TCEP and IAA, respectively, improves The samples are ready to be submitted to the Triethylammonium bicarbonate buffer 1 M, suitable for HPLC, LiChropur; CAS Number: 15715-58-9; Synonyms: Triethylammonium hydrogen carbonate buffer; find Supelco-18597 MSDS, related peer-reviewed papers, technical documents, similar products & more at Sigma-Aldrich preparation while others need to be prepared just before use as needed; therefore, (E) Integrated areas for specific extracted ions from one sample peptide. Hide. require fractionation, prepare larger volumes of the elution solutions to accommodate Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature This saves time and money when coming up against roadblocks with separation development as, once all of the usual buffers have been tried, attention turns to changing the column chemistry, which may not be necessary. Sample preparation can be performed in 2 alternative ways using, Microcentrifuge polypropylene tubesMicrotip probe sonicator or nuclease (e.g., Thermo Scientific Pierce Universal Nucleasefor protein stabilizers glycerol, PEG, which severely interfere with MS analysis. even at low concentrations; some of these agents may interfere with specific step(s) Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Reducing reagent: 30 mg/mL TCEP (~100 mM, Sigma C4706) or 15 mg/mL DTT (Sigma D0632 ) in digestion buffer. Warm the Cell Lysis Buffer and Digestion Buffers to room temperature before use. J Biomolecular Techniques.11:74-86. below). limits vary considerably based on application and instrumentation, Low protein yield following lysis and protein extraction procedure, Estimate protein concentration using BCA assay, Lyophilized/dried peptide samples were not completely solubilized before sample loading in the gel; during this step you must prevent the gels/wells from drying. A second protocol, included, provides instructions for digesting molecular Disulfide bonds should be reduced prior to the start of the FASP Protein Digestion Carefully remove acetone without dislodging the protein pellet. filter,vortex 1 min, and incubate at 37C for 2 hours.8. and clean-up for peptide sequencing. proteolysis at 37C, all the protein will be solubilized. (2001). Ammonium bicarbonate is an irritant to the skin, eyes and respiratory system. Combining the search results This method yields more protein lysate from cultured cells, is highly reproducible, is scalable from 10g to 5mg, is simpler and faster than FASP, has no risk of carbamylation by urea, and results in higher protein identification rates than other popular standard sample preparation methods (Figure 2 and Table 2). Add 200L of Urea Sample Solution to the Spin Filter, cap the filter, vortex and hemoglobin in red blood cells, albumin Alkylation is optional, but highly recommended. FASP columns) or by acetone precipitation. Volatile salts are the only salts compatible with MS. Aqueous solutions of ammonium bicarbonate (0.01 - 0.1 M) have pH around 8, the optimal pH for trypsin activity. of Alkylation Buffer to the tube. The Thermo Scientific Pierce C18 Pipette Tips enable fast and efficient capture, concentration, Prepare 800 mL of distilled water in a suitable container. protocol for best results. Mix However, this has been shown not to be the case in many examples and has come to be known as wrong way round ionisation [7,8]. Alternatively, use Pierce Universal Nuclease for Cell Lysis(P/N determine (in collaboration with a statistician) an optimal/required number of replicates Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vialContaining 20g trypsin and incubate at room temperature Speicher, K.D., et al. Mass Spectrom. Analysis of medium and low abundant proteins is extremely difficult/impossible in Bereman, M.S., Egertson, J.D., MacCoss, M.J. (2011). For the best experience on our site, be sure to turn on Javascript in your browser. The data in this article were previously presented at the 2013 American Society for Mass Spectrometry annual meeting in a poster titled: A Versatile Sample Preparation Procedure for Shotgun Proteomic Analyses of Complex Samples by Mass Spectrometry. 1. can be used as an internal standard. the downstream application. It possesses a strong ammoniacal smell, and on digestion with alcohol, the carbamate is dissolved leaving a residue of ammonium bicarbonate.[3]. 4. This is especially useful in the analysis of peptides and proteins and typically 5mM of medronic acid can be added to buffered mobile phase (ammonium acetate for example) to provide highly-effective deactivation, resulting in improved peak shapes, detector sensitivity, and quantitative reproducibility. Mixand incubate at room temperature for 20 minutes protected from light. Click here to see all available distributors, Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6), Change the value in the textbox above to scale the recipe volume, Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) Preparation and Recipe, PBS (Phosphate Buffered Saline) (1X, pH 7.4), BES-Buffered Saline (2X) (0.05 M, pH 6.95), Citrate-Phosphate Buffer (0.15 M, pH 5.0), Citrate-Phosphate Buffer (110 mM, pH 5.6), EBSS (magnesium, calcium, phenol red) (pH 7.0), Glycine-Sodium Hydroxide Buffer (0.08 M, pH 10), Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0), Penicillin/Streptomycin/Chloramphenicol Antibiotic Mix, Yeast Two Hybrid (Y2H) Media, Amino Acid Dropout Mixes, Sodium Carbonate Transfer Buffer (40x, pH 9.5), https://www.aatbio.com/resources/buffer-preparations-and-recipes/carbonate-bicarbonate-buffer-ph-9-2-to-10-6, Adjust the molarity of the solution by using the slider below, Adjust the pH of the solution by using the slider below. Sequences of the five peptides that result from the Digestion Indicator, and coefficients of variation (CV) for triplicate samples processed using the Pierce protocol (Part No. Protein extracts can be separated from these low MW components by filtration using as 35% for hydrophilic peptides. Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT Determine the peptide concentration in the samples using Pierce QuantitativeColorimetric Numerous experiments consistently found that these faster, high-resolution instruments identify many long, higher charged peptides with missed cleavages that are not detected on lower-resolution, slower mass spectrometers. Product shelf life If greater than amount of reagents (DTT, IAA, Lys-C and trypsin). For example, to test reproducibility of our optimized method, we processed and analyzed quadruplicate samples of a HeLa cell culture using the Pierce protocol, spiking the Digestion Indicator into each lysate after the initial lysis step (same method as for Table 3). Note: Some of the solutions required for the In-Gel Tryptic Digestion Kit require occasional Use a spot picker or scalpel to excise protein band of interest from 1D or 2D gel. This protocol reproducibly yields high-quality peptide samples for LC-MS/MS analysis that provide high rates of protein identification as a result of efficient and selective protein extraction, reduction, alkylation, and digestion (Table 3). Add 4l of trypsin (2g, enzyme-to-substrate ratio = 1:50) to the sample. desalting and elution of peptides. Iodoacetamide Solution by adding 100 L Urea Sample Solution to one tube of Iodoacetamide digestions for protein identifications in proteome studies. Culture cells to harvest at least 100g of protein. Warm the Cell Lysis Buffer and Digestion Buffer provided with Pierce kit to roomtemperature on an Agilent protein chip, which are available in the MRC. or more samples representing different conditions (groups) - e.g. Mix 96.4 ml of solution I with 3.6 ml of solution II. The size of a band to be excised from a gel should not exceed these Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N Oxalic Acid - C. 2. Cell Lysis, P/N. or 100L tip, respectively. Comments having links would not be published. dispense and aspirate sample for 3-10 cycles. protein stains and the Additional Information Section for alternative destaining procedures. The Destaining Solution may be stored at 4C for 2 months. incubateovernight at 37C.6. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample. Seppro Ammonium Bicarbonate Buffer. Store buffers at 4C. Centrifuge Product is shipped on dry ice. For maximum efficiency, This solution may be stored at -20C for 2 months without in the presence of highly abundant proteins (e.g. Acetonitrile (ACN), LC-MS Grade (Product No. Ammonium hydrogen carbonate is an excellent buffer for use at high-pH with good buffering capacity over pH 8-11 and possibly wider at higher ionic strength. Short-term health effects may occur immediately or shortly after exposure to ammonium bicarbonate. Aspirate up to 10L of sample (prepared in Step 2) into the C18 tip. for statistical validation of results.
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