ammonium bicarbonate buffer preparation

Mix of 2 10. Methods. 7. They were once produced commercially, formerly known as sal volatile or salt of hartshorn. Finally, 500ng samples were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive mass spectrometer. Standard Buffer Solutions are solutions of standard pH. Lahm, H.W. 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). For the best experience on our site, be sure to turn on Javascript in your browser. Cold (-20C) acetone, a volume four times that of the protein samples to be precipitated, Centrifuge tube, made of acetone-compatible polypropylene and able to hold five times The compound has many names, reflecting its long history. be prepared three times with this kit. the number of identified proteins relative to unfractionated samples. This analysis indicated <10% missed cleavages. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge hemoglobin in red blood cells, albumin Because sample preparation is the most problematic area of MS-based proteome analysis, it is important to have robust, reproducible methods that can be easily adopted by novice and expert MS labs alike. Add 200 L of Urea Sample Solution to the Spin Filter and 1. centrifuge at 14,000 Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Static modifications included carbamidomethyl (C) and dynamic modifications included oxidation (M). Centrifuge the Spin Filter at Analysis of medium and low abundant proteins is extremely difficult/impossible in Adjust the pH, if necessary. Ammonium Bicarbonate pK a 3 10.30 9.3-11.3 NH 4HCO 3 HCO 3- CO 3-2 0.79 g HCOOH or NH 4OH Wet tip by aspirating 100L of 50% ACN in water and then discarding solvent. Wash buffer: 0.1% acetic acid in water. Selective depletion of abundant proteins from protein extracts (to Discard the flow-through from the collection tube. J Biomolecular Techniques.11:74-86. Do not exceed the recommended centrifugation speeds because this may damage the column solution in single-use volumes at -80Cfor long-term storage.5. Product Usage Information. It was obtained by the dry distillation of nitrogenous organic matter such as hair, horn, leather. To assess the scalability of the reduction, alkylation, digestion steps in the sample preparation protocol, we processed five different amounts of HeLa cell lysate (10, 50, 100, 200 and 5000g) using the method. 84840). with 20L of the supplied Trypsin Storage Solution. Disulfide bonds should be reduced prior to the start of the FASP Protein Digestion Repeat this step once. settings for your system, Verify LC-MS system performance with the Thermo Scientific Pierce HeLa Digest Protein Dilute stock 10-fold by adding All articles and SOPs are written by Ankur Choudhary. Add 100 L of 50 mM Ammonium Bicarbonate Solution 9. provided with the FASP Kit to with MS analysis and result in sample loss from nonspecific adsorption. For binding to C18 reversed-phase sorbents, a sample must be free of excess organic Ammonium Bicarbonate, 1M (for Molecular Serology) Y Ammonium Bicarbonate, 50mM (for Molecular Serology) Y Acetic Acid, 5% (for Molecular Serology) Y Acetic Acid, 0.03% (for Molecular Serology) N Acetonitrile with 0.1% FA (for Molecular Serology) Y ATL Buffer Y BCA Kit Reagent A (for Molecular Serology) Y BCA Kit Reagent B (for Molecular Serology) Y Discard the flow-through from the collection tube3. Repeat Buffer. J. (mBIO) core (x8-3743) if you are unsure about statistical requirements for an experiment. dimensions: 1mm X 1mm X 5mm. Add 9.274 g of Sodium carbonate (anhydrous) to the solution. concentrate digest on C18 sample prep device (Product No. 100%acetone to sample. (Sigma, P/N T7408-100ml). Add 75 L Digestion Solution (enzyme-to-protein ratio 10. Repeat Figure 2: Medronic (Methylenediphosphonic) acid (pKa 1.27). once. 1) When preparing an ammonium acetate 5mM buffer solution with pH=3.3, which is better to use to adjust the pH? 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). Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of100% preparation while others need to be prepared just before use as needed; therefore, Do not store high-pH Cool the lysate on ice for 5 minutes, spin down. solvents such as acetonitrile (ACN) or methanol. Centrifuge at 14,000 x g for 25 min. . Place protein sample in acetone-compatible tube. producing complete and accurate digest for dependable mass spectrometric (MS) analysis. Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the Search fractionation, high-pH reversed-phase fractions do not require an additional desalting an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. The final concentration pipette up and down to dissolve the contents of the tube. Benchtop centrifuge capable of 14,000 x g. Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vialContaining 20g trypsin and incubate at room temperature analysis. some of them may, as denaturing agents, interfere with the proteolytic digestion step Ammonium hydrogen carbonate has been described as an excellent buffer for the analysis of basic drugs by HPLC-MS (10). In addition to ammonium bicarbonate, this material contains ammonium carbamate (NH4CO2NH2), and ammonium carbonate ((NH4)2CO3). protein pellet. Electrophoresis22:2058-65. Warm the Cell Lysis Buffer and Digestion Buffers provided with Pierce kit to roomtemperature Diagram of the developed protocol. Transfer the alkylated protein sample (step C9) into the Spin Filter. the Spin Filter and centrifuge at 14,000 x g for 10 min. 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). Determine the peptide concentration in the samples using Pierce Quantitative ColorimetricPeptide anyunused IAA solution.9. 12. We have noted, along with other literature reports [3] that the addition of the perfluorinated acids to the sample diluent can have a marked effect on the peak shape, and sometimes on the retention time stability of the resulting chromatography. Centrifuge lysate at 16,000 g for 10 minutes at 4C. This Agilent run will Protein alkylation in the presence/absence of thiourea in proteome analysis: 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. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protocol for best results. Do not introduce air through the membrane Subsequently, 100g amounts of each replicate lysate were processed by the respective protocol. When required, prepare trypsin stock solution by hydrating the lyophilized trypsin Discard the flow-through from the collection tube. Hide. Urea Sample Solution FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250Kit Contents (sufficient for processing 8 samples): 1. Store FASP Protein Digestion Kit materials at room temperature. Seppro Ammonium Bicarbonate Buffer. overnight with shaking. analysis, peptides in each high-pH fraction are further separated using a low-pH gradient, Usually, they are not necessary for sample processing From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by each method. sensitivity and high-quality spectra. Store any remaining trypsin Pierce Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840Kit Contents (sufficient Sodium Carbonate - Sodium Bicarbonate Buffer Preparation, pH 9.2-10.8 Buffer Preparation Formulas and Equations Choosing the Right Biological Buffer Choose a buffer based on your pH requirements as well as the pKa, a measure of acid strength that accounts for pH, concentration, and temperature. 6. method using acetone is presented here. Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography, M. Shibue, C.T. Standard (Product No. concentration). 1:100) and vortex for 1 Therefore, they must be removed before LC/MS analysis at appropriate processing steps. Resulting lysate samples (200g in 200L of Lysis Buffer) were spiked with 2g Digestion Indicator and processed through remaining steps of the Pierce protocol. Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l Processing/preparation of protein extracts for LC/MS analysis include trypsin digestion. 84841), to monitor and compare the efficiency of sample prep experiments. Incubate sample in the dark at room temperature 2. per 1ml ofcell lysate and incubate at room temperature for 15 minutes.6. S is the centrifuge speed in rpm. Before trypsin 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. Cool the lysate on ice for 5 minutes, spin down..5. As a very approximate rule of thumb follow these guidelines; Remember that trifluoroacetic acid (TFA) is a strong ion pairing reagent and may severely restrict the detector sensitivity in positive ion mode, because the ion pair is strong enough to survive as a neutral complex with the analyte during liberation into the gas phase. g for 10min. Add 100 L of Urea Sample Solution to the Spin Filter and 7. centrifuge at 14,000 in this form at -20C for > 1 year without significant loss in activity. After alkylation with IAA, immediately add 690l (6 volumes) of pre-chilled (-20C) In addition, Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide Click here to see all available distributors, Change the value in the textbox above to scale the recipe volume, Ammonium Bicarbonate (50 mM, pH 7.8) Preparation and Recipe, PBS (Phosphate Buffered Saline) (1X, pH 7.4), BES-Buffered Saline (2X) (0.05 M, pH 6.95), Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6), 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/ammonium-bicarbonate-50-mm-ph-7-8. Store each aliquot at -20C in a nonfrost-free 23225) or Thermo Scientific Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Part No. Rinse the tip by aspirating 10L of 0.1% TFA/5% ACN and discarding solvent. Repeat this step twice. 2. at 37C for 2 hours.4. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. centrifugeat 14,000 x g for 12 min. If you have used Protein Discoverys UPX Universal Protein Extraction Kit or YPX The reagents required for the preparation of standard buffer solutions are described here. 3. The FASP Protein Digestion Kit is compatible with whole proteome extracts and other Reduction and Alkylation (Optional) Prepare new 5mM TCEP solution by diluting 10L of 0.5M TCEP in 1mL of 100mM ammonium bicarbonate. Bear in mind the UV contribution of the additive when working at low UV wavelengths (<220nm) and be smart with UV detector settings to avoid sloping baselines [1,2]. with a proteolytic enzyme (usually trypsin) and generated peptide mix is subjected up thecell clumpsand gently vortex sample to mix.3. Each cell suspension was sonicated on ice for 20 seconds (pulse time 5 sec, pulse off time 5 sec, output level 2) using a Misonix 3000 Sonicator. from at least 20ng of protein containing at least 0.5ng of each singular peptide product. only the number of cycles necessary for the application. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the require fractionation, prepare larger volumes of the elution solutions to accommodate x. Store the remaining components Incubate sample at 37C for 4 hours or at 30C Gel Electrophoresis. Add 200L of Urea Sample Solution to the Spin Filter, cap the filter, vortex and Vortex the tube until all To avoid inaccurate volumetric dispensing, do not use Pierce C18 Pipette Tips for the protein pellet.11. If necessary, receiver tubes used for the final collection may be Adjust sample to 0.1-1.0% TFA using 2.5% TFA. There is no absolute single best way to lyse cells and extract proteins. 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. Mixand incubate at room temperature for 20 minutes protected from light. Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics Load 300L of the appropriate elution the presence of highly abundant proteins (e.g. Proteomics 11:2931-5. that inactivate and protect the enzyme from autodigestion. The carbonate/bicarbonate anion system has two pK values, one at 6.4 and one at 10.3. Place the column into a new 2.0mLsample tube. thus reducing the overall sample complexity and improving the ability to identify Peptide Assay (Product No. Buffer pKa and pH Range Values For preparation of . 1:100) and vortex for 1 min. 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 . Product is shipped on dry ice. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity Precipitation has an advantage over dialysis or desalting methods in that it enables IntroductionThe Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells enables during any portion of the procedure for optimum flow and peptide recovery. of MS instrument including its ion optics. Cool the lysate on ice for 5 minutes, spin down.5. Before use, leave any home made gels overnight on the bench IntroductionThe Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells enables No. Digestion Buffer may be stored at 4C for 2 months. If local exhaust ventilation or enclosure is not used, respirators are necessary. Sample Preparation. Mix 80mg of ammonium bicarbonate with 20mL of acetonitrile (ACN) and 20mL of ultrapure Note: The centrifugation times may need adjustment keep it short but long enough to let It Medronic Acid (Figure 2) can be used as a very useful alternative to EDTA with LCMS analysis and has been shown to produce much lower degrees of ion suppression. also provided with the FASP Kit. add 1ml of 1M TEAB to 19ml of ultrapure water, mix. byshearing DNA. Again, MSA produces altered selectivity to TFA and there are reports that addition of MSA to TFA based eluent systems in HILIC mode can be used to tune the selectivity in this separation mode [6]. generated by the individual fractions improves protein sequence coverage and increases Ammonia Buffer pH 9.5: Dissolve 33.5 g of ammonium chloride in ISO ml of water, and 42 ml of 10M ammonia and dilute with water to 250 ml. Cell Lysis, P/N. Mix and dissolve the solution by pipetting it up and down Shrink gel pieces by adding 50L of acetonitrile. Elution buffer: 75% acetonitrile, 5% acetic acid, 20% water. stopping additional enzymatic activity. Add 100l of Digestion Buffer provided with Pierce kit6. Shevchenko, A. and Shevchenko, A. pipette upand down to dissolve the contents of the tube. The maximum loading capacity of one FASP Protein Digestion Kit is 0.4 mg protein in Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l Finally, 500ng samples were analyzed by LC-FT MS/IT MS2 CID on a Thermo Scientific Orbitrap Elite mass spectrometer. 88700), Protein assay kit (e.g., Thermo Scientific BCA Protein Assay Kit, P/N 23227), Chilled (-20C) 100% acetone and 90% acetone, Vacuum concentrator (e.g., Thermo Scientific SpeedVac Vacuum Concentrator), Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of100% protein extracts are then dissolved and trypsin digested in an appropriate buffer. 10 samples are being digested simultaneously, increase the volume of stock accordingly. Add 200 L 2. . LC/MS analys is used for identification of proteins in analyzed samples and mapping of 84840). Commonly used for various immunoassay applications and for many protein and antibody conjugation procedures, including sandwich ELISA, which require experimental surface coatings. as well. 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 IAA is ~500mM. bands. Figure 5. The samples are ready to be submitted to the Glycine Buffer Solution: Mix 42 g of sodium bicarbonate and 50 g of potassium bicarbonate with 180 ml of water and add a solution containing 37.5 g of glycine and IS ml of strong ammonia in 180 ml of water. For our compounds, pH 11 seems to be optimal because we cannot reduce the aqueous composition down to 10 mM (pH. Dilute 7L of the 5X stock solution with 28L of Digestion Buffer Discard This mode of ionisation is reported to be driven by process such as; Therefore, It is incumbent upon us to explore separations involving basic analytes at high pH to gain alternative selectivity, even if this appears to be counterintuitive to theory. If greater than Cool the sample to room temperature for 10 minutes, spin down.7. Well, this procedure is good enough for a rough screening solution- call it a quick and dirty method. vialContaining 20g trypsin and incubate at room temperature for 5 minutes. 13. Transfer 15 times. Peptide fragments with one missed cut are common and should be taken into 4. Salts/Buffers decrease sensitivity, greatly complicate MS analysis, and damage essential elements Note: Some of the solutions required for the In-Gel Tryptic Digestion Kit require occasional Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the They have been widely reported in literature as long ago as 1996 [3 - 5]. desaltingproducts are available for performing such buffer exchanges with small or the high sensitivity and mass accuracy. solution in single-use volumes at -80C.9. 4. facilityfor LC/MS analysis. pipette upand down to dissolve the contents of the tube. to remove the (volatile) Digestion Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. 89870), Note: Limits vary considerably based on application and instrumentation, 1. Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature Salts, buffers, other small hydrophilic Kit toone tube of Urea, also provided with the FASP Kit. The required amount of digested protein in submitted samples is at least 0.2g JavaScript seems to be disabled in your browser. of IAA is ~500mM. Add 200L of Urea Sample Solution to the Spin Filter, cap the filter, vortex and gels. The final concentration Sample is now ready for liquid chromatographic separation and electrospray ionization Swell A Thermo Scientific EASY-nLC 1000 HPLC system and Thermo Scientific EASYSpray Source with Thermo Scientific EasySpray Column (25cm x 75m i.d., PepMap C18) was used to separate peptides (500ng) with a 30% acetonitrile gradient in 0.1% formic acid over 100-140min at a flow rate of 300nL/min. When using 10g of cell lysate, Aftercentrifugation Centrifuge lysate at 16,000 g for 10 minutes at 4C.7. Remove the protective white tip from the bottom of the column and discard. Warm and equilibrate the Pierce Digestion Indicator to room temperature. 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. Make a 10X Table 1. They are used for reference purposes in pH measurements and for carrying out many pharmacopoeial tests which require adjustments to or maintenance of a specified pH. Mix and To convert from revolutions per minute (rpm) to g, use the following formula: where g is the relative centrifugal force, R is the rotor radius in centimeters, and Shevchenko, A., et al. Compare Product No. Mass spectrometry (MS) has become a prominent technique in biological research for the identification, characterization, and quantification of proteins (Ref. This buffer calculator provides an easy-to-use tool to calculate buffer molarity and prepare buffer solutions using the formula weight of the reagent and your desired volume (L, mL, or L) and concentration (M, mM, or nM). the manufacturers protocol.14. After alkylation with IAA, immediately add 100l of Urea Sample Solution and proceed desalting and elution of peptides. incubateovernight at 37C.6. Note: The recommended amount of trypsin used per digest is 100ng (see protocol). Working Solution an additional four-fold with Digestion Buffer. 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. tubewith an empty pipette tip. Buffers pKa range . in the Spin Filter and centrifuge at 14,000 x, Add 200 L of Urea Sample Solution to the Spin Filter and 2. centrifuge at 14,000 Discard the flow-through from the collection tube. for optimum tip-to-pipettor seal and sample aspiration. If using nuclease, add 25 units of nuclease is important to dissolve as much protein as possible; water bath sonicationmay facilitate Breathing ammonium bicarbonate can irritate the nose, throat and lungs causing coughing, wheezing and/or shortness of breath. The Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit provides Purified protein extracts are then dissolved and trypsin digested in an appropriate Compare this to the use of ammonium acetate or formate buffers at low pH where the buffering ranges of the ammonium species and the format or acetate are several pH units apart (see Table 1). Contaminants may be introduced at several steps during sample preparation. Trypsin is a serine protease that specifically cleaves peptide bonds at the carboxyl The ion-pair tends to dissociate within the ESI source, giving rise the corresponding charged analyte in the gas phase. If using nuclease, add 25 units of nuclease Purified A trypsin fragment For the Pierce protocol, HeLa cell lysate (100g) with digestion indicator (1%, w/w) was reduced with 10mM DTT for 45 minutes at 50C and alkylated with 50mM iodoacetamide for 20 minutes in dark at RT. One disadvantage of protein precipitation is that proteins might denature, making Add 100l of ultrapure water to the tube and gentlypipette We recommend the preparation for just 4 . Shortly before use (Step C.3) dilute 1L of Trypsin Working Solution with 9L of Digestion 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. Rinse the tip by aspirating 100L of 0.1% TFA/5% ACN and discarding solvent. out Universal Sample preparation as described by Wisniewski, Zoubman, Nagaraj and Store high-pH buffers in polypropylene tubes at room temperature. Unfortunately, when ammonium bicarbonate was used as a buffer reagent in electrospray ionization analysis, proteins formed higher charge states, indicative of protein denaturation . Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature Therefore, we developed and optimized the double-digestion LysC-trypsin protocol until it consistently resulted in less than 10% missed cleavages on Thermo Scientific Q Exactive and Orbitrap Elite instruments. It is used in, for example, Swedish "drmmar" biscuits and Danish "brunkager" Christmas biscuits, and German Lebkuchen. primarily for MS applications, they may be used for applications such as peptide concentration Volatile Buffer Structure pK a Buffer Range Triuroacetic acid CF 3CO 2H 0.5 3.8-5.8 Formic acid HCO 2H3.8 Ammonium formate HCO 2NH 4 3.8 2.8-4.8 Acetic acid CH 3CO 2H4.8 Ammonium acetate CH 3CO 2NH 4 4.8 3.8-5.8 4-Methylmorpholine OC 4H 8N(CH 3) 8.4 7.4-9.4 Ammonium bicarbonate NH 4CO 3H 6.3/9.2/10.3 6.8-11.3 Ammonium acetate . Centrifuge A variety of Thermo Scientific dialysis and reproducible processing of cultured mammalian cells for proteomic mass spectrometry Many users recommend that columns used with TFA are dedicated to separations using this eluent additive. Add 30L of Reducing Buffer to the tube . however, the procedure may be used for 10-200g of cell lysate protein with an appropriate Discard Matrix-assisted laser desorption ionization (MALDI-) and electrospray ionization (ESI-) 4. Native, an optimized fractionation protocol and reagents to increase the number of proteins 8. It is commonly used as an inexpensive nitrogen fertilizer in China, but is now being phased out in favor of urea for quality and stability. diluted with digestion buffer, Ensure gel slice has been completely destained, Concentration or detection limits of application, Clean-up digest with C18 sample prep device, Dry sample and resuspend in 10L or 100L of 0.1-1.0% TFA, Ensure that air is not drawn into the tip and that sorbent does not dry during sample Load 300L of the sample solutiononto However, if protein band contains significantly less than ~20ng If sample is reduced and alkylated before or during electrophoresis, it may measuring volume. The final prepared samples are ready for direct MS analysis or other downstream applications, including peptide fractionation, mass-tag labeling, or phosphopeptide enrichment.
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