Tca precipitation protocol. Add 1 volume of TCA stock to 4 volumes of protein sample.

Tca precipitation protocol When using TCA You must know, that in some cases easy to obtain hydrolised products (big and small peptides), so after addition TCA, add gaseous NH3 to neutralise solution. (preparation of 100% TCA: 454ml H2O/kg TCA. I found a precipitation protocol with TCA/Acetone (https://www. Add TCA to the combined reaction mixture in a 1:4 (vol/vol) ratio and incubate the mixture at 4 °C on a rotator overnight. , 32 P-dATP or 32 P-UTP). Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. incubate on ice for 30 min 7. This item requires a subscription to Cold Spring Harbor Protocols. Follow the guidelines to avoid keratin contamination and adjust pH if needed. in 1. We systematically It may be concluded from our study that the best conditions for equine serum albumin and IgG removal by the TCA protein precipitation method are use of 10% TCA in acetone as the precipitation solution and precipitation by dilution of serum in the precipitation solution at a ratio 1:5 and incubation at −20°C for 90 min. 2 and 3. TCA/Acetone is routinely used for precipitatin of proteins. So what we thought earlier to be DNA crashing out, is actually protein that doesn't seem to like 4xSDS. Surprisingly little is known of the mechanism. It is important to remove all the TCA because otherwise it will acidify the sample buffer, making it go green. Another problem I face is after TCA precipitation, when I try to boil that precipitate with 2X sample Jun 15, 2022 · However, the TCA/acetone procedure led to a partially insoluble pellet, and a considerably lower number of proteins were in the following identified, compared to when only using acetone-precipitation (20% vs. TCA Precipitation ­ add TCA to a final concentration of 25%, vortex ­ put on ice for 30 min w/ frequent vortexing ­ spin at 14K, 4ºC for 30 min ­ remove supernatant and add 1mL ice cold 100% acetone, vortex ­ spin at 14K, 4ºC, for 5 min ­ remove supernatant and add 1mL ice cold 100% acetone, vortex Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2-D electrophoresis, and is more effective than either TCA or acetone alone. Below is my protocol and does anyone have good idea to dissolve pellet (at step 8)? Many thanks to all of you! 1. Trichloroacetic acid (TCA)/acetone precipitation, as described here, is often used for this purpose. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. 3 OD units (Δ15 µl) run out on gel: Affinity Purification and/or Pulldown: 30 ODs of lysed cells: Bind, elute, and prepare proteins as described in steps 3. In the protocol, nucleic acid synthesis reactions are carried out in the presence of a radiolabeled nucleotide (e. As expected, no SseC protein could be Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2-D electrophoresis, and is more effective than either TCA or acetone alone. For low concentrations, leave longer. Grind plant tissue in liquid nitrogen. To overcome the limitations. 1. [2], Huang et al. Remove the supernatant, leaving the protein pellet intact. TCA precipitation denatures the protein, so it should not be used if the protein … Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2-trichloroacetic acid (TCA). However, most of the Deoxycholate+TCA precipitation protocols seem to The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. Link and I am trying to evaluate by western blotting proteins secreted by cells to the culture medium (in cell cultures). TCA protein precipitation (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology) Procedures and stock solutions The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. Maintain 4C in dark bottle). Stock Solutions: 100% (w/v) Trichloroacetic acid (TCA) recipe: dissolve 500g TCA (as shipped) into 350 ml dH 2 O, store at RT. Table 8 lists some of the precipitation techniques that can be used. 10 %). harvest yeast cells corresponding to an OD 600 = 1-2 I ran sucrose gradient of 5-35% (w/w) and proceed with TCA precipitation. acid (TCA) precipitation is frequently used to remove interfering compounds. One more thing-there is a note on file (im doing this on behalf of someone not here currently that they used 50mL of supernatant concentrated down to 100uL to detect Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2-D electrophoresis, and is more effective than either TCA or acetone alone. TCA/Acetone Precipitation is one of the most common Precipitation methods. This method is useful for obtaining prote The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. One more thing-there is a note on file (im doing this on behalf of someone not here currently that they used 50mL of supernatant concentrated down to 100uL to detect TCA-DOC For very low protein concentration To one volume of protein solution, add 1/100 vol. 45 min TCA/DOC precipitation - (Dec/06/2005 ) Hi To precipitate proteins in minimal growth media for secretome analysis, I added Na DOC to 0. add 230 μl 80 % TCA to protein sample (min. May 13, 2002 · If a small amount of protein is to be precipitated (less than a few micrograms), add Insulin as a carrier protein (10 micrograms of Sigma insulin, I-5500, per sample works well). Method 1 used a short incubation in 80% TCA at room temperature, as liver, river biofilm, soil) after TCA precipitation and solvent purification. However some protocols refer to using Acetone alone. 61. Make up to 100 mL with milliQ water. edu Nov 1, 2020 · The three chosen protocols were the precipitation by M/C with UH cycles, acetone using the standard protocol, and TCA-acetone with FT and UH cycles without 2-ME, with the inclusion of a NaOH pre-solubilization step in the three protocols. Alternative protocols have been proposed, using acetone alone, methanol/chloroform , ethanol , or a combination of pyrogallol red, molybdate, and methanol . The thorough RSM analysis clearly shows in Figure 1 that the stationary point of TCA-mediated protein precipitation is a saddle point. Jan 16, 2014 · Therefore, this protocol is different from the previously reported TCA/acetone precipitation 12,13,21 and phenol extraction protocols 13,22,56 . of TCA solution (125ul in 500ul sample). Optional step. 6. Vortex then incubate on ice for 30 minutes. Materials Protein solution Ice Centrifuge 2X SDS buffer TCA Procedure protein precipitation 1. Mar 31, 2016 · The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield. 2. TCA is added to the extract to a final concentration of 10–20% and the proteins are allowed to precipitate on ice for 30 min (46). its. i. In this study, we examine the mechanism of the TCA-induced protein precipitation reaction. Prepare for Western blot by TCA precipitation as described in step 2. Similarly, Vincent et al. 8 kDa (Fig. Carefully remove sup and washed with 500 ul acetone. Protocols. Dec 17, 2018 · Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. There are slight differences in the protocol if when used for other purposes. 1A) could be precipitated in the CS fraction and in the SD fraction of CREA0300 and CREA0300(pLRC5T). 8, 8 M urea, 5% w/v SDS, 1 mM EDTA, 100 mM DTT, bromophenol blue; stored at –20 deg w/o DTT; DTT is added freshly from 1 M stock) cell lysis and protein precipitation: 1. Protein Chem 16 , 291-7 (1997) show that precipitation vs TCA concentration is a U shaped curve with optimum ppt at roughly 15% TCA and May 17, 2023 · Protein extraction with TCA (trichloroacetic acid) and acetone is a widely used method to precipitate proteins from biological samples. [3] and Song et al. Section 1. Incubate 30 min on ice. Incubate for 30 min on ice 9. Lanes correspond to: (1) marker, (2) urine starting material, (3) TCA/acetone precipitation supernatant, (4) TCA precipitate, (5) urine supernatant after 3 h centrifugation at 200,000 RCF, (6) protein pellet after ultracentrifugation of 5 mL urine, (7) urine filtrate after ultrafiltration through 5 kd MWCO, and (8) urine retentate after TCA precipitation - What is the final concentration of TCA ? (Apr/18/2002 ) Hi, I have read some protocols that use 10% but some use 20%. Add 1/10 of TCA 100% vortex and let sit ON at 4C. 5M Na2EDTA. g. transfer supernatant to a new Eppendorf tube 4. 07% 2-mercaptoethanol or 20 mM DTT. 6. Protein precipitation methods or protocols are used for the concentration of diluted proteins in solution. Precipitated The efficacy of the novel protocol was compared to that of the standard protocols for protein precipitation employing TCA and acetone (Fig. The goal when using this methods is to purify and concentrate contaminated proteins or proteins dissolved in various matrices, buffers, detergents or from natural sources, such as blood, urine or other biofluids. Apr 20, 2015 · Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Δ0. 02% (4 C, 30 min) and then TCA to 10% (4 C, overnight). HCl [pH 8. TCA precipitation TCA (trichloroacetic acid) is a very effective protein precipitant. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. TCA precipitation, what is the best solution to dissolve followed by Mass spectr - (reply: 1) Need to increase protein precipitation yield w/ TCA precipitation protocol - (reply: 3) Hard to dissolve pelleted protein after TCA precipitation - Urgent!! Feb 1, 2007 · In past, organic solvent mediated precipitation based protocols such as acetone [3][4][5], To address this, TCA-A was compared with another acetone-based protocol (TCA-B) or a phenol (Phe May 17, 2023 · Protein extraction with TCA (trichloroacetic acid) and acetone is a widely used method to precipitate proteins from biological samples. Protocols > TCA acetone precipitation method TCA/Acetone protein precipitation method for plant proteins. Add 1 volume of TCA stock to 4 volumes of protein sample. Procedure. Transfer supernatant and TCA to 40 ml Avanti tubes, centrifuge 15 min at 20. Proteins were conjugated with the different CyDyes after extraction with the TCA-DOC, 24 h, post-confluence (Cy2) (A), TCA-NLS-THF, 48 h, post-confluence (Cy3) (B) and TCA-DOC, 48 h post-confluence (Cy5) (C) induction conditions and precipitation methods and resolved by 2D-DIGE from one control cell line. Add 1 ml of ice-cold ethanol Footnote 3 and centrifuge briefly. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. TCA Precipitation with Acetone Wash . Each individual spot is circled and was National Center for Biotechnology Information Nov 14, 2018 · TCA precipitation is used to precipitate proteins when measuring the incorporation of radioactive precursors in metabolic studies and can be used to prepare samples for liquid chromatography tandem mass spectrometry [5, 14]. The precipitation and rinsing solutions must be cold when used. 10 mM Tris. By the PRMM protocol, SseC migrating to 52. TCA-induced protein precipitation curves are U-shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. 3) May 7, 2010 · Hello, I am precipitating some fractions that I believe contain protein. • Data highlight the efficiency of ethanol/HCl purification for TCA-precipitated proteins. Protein purification is more efficient when TCA Jan 1, 2011 · Add 1 volume of 10% TCA Footnote 2 to the sample (i. Linse. Continue grinding with 10% TCA solution in acetone (ice-cold). Follow the steps for sample preparation, centrifugation, washing and resuspension. This protocol is for this application of TCA precipitation. 29% proteome coverage for protocol 8 = TCA/acetone, vs protocol 7 = acetone; Fig. This document provides a 10-step protocol for precipitating proteins from cell lysates using trichloroacetic acid (TCA) and acetone. Also, it may be possible to concentrate a dilute large-volume sample by scaling up the precipitation step while maintaining or reducing the volume of working reagent used to dissolve and assay the pellet. 18 reported that this protocol resulted in improved 2-DE gels for mature grape berry clusters. Protein Chem 16 , 291-7 (1997) show that precipitation vs TCA concentration is a U shaped curve with optimum ppt at roughly 15% TCA and The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. Precipitation yields depend on complexity of your sample,concentration and duration The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. 5 M NH4OAc and 200 uL of 0. Trichloroacetic acid (TCA) is very nasty! precipitation methods for direct use in the Microplate Protocols, but this has not been tested. 3: Western Blot of Purified Proteins: Approximately 1-2 ODs (if prepared as in step 3. But someone said that it is really hard to dissolve pelleted protein with 1x sample buffer for SDS-PAGE. Aug 1, 2011 · Trichloroacetic acid (TCA) precipitation with acetone washes was performed to further concentrate the samples as described previously (TCA) following standard protocols 40. For 100 mL add 1 mL 1 M Tris. Here we detail the most classical assay used to precipitate proteins of bacterial culture supernatant based on TCA precipitation ; it is used widely in secretion system studies. Learn how to prepare samples for in-gel digestion or SDS-PAGE using TCA and DOC. In this protocol, we use 0. Spin 10min 4C in microfuge at maximum rpm (15000g). I saw no pellet after the first spin. And what is the rational So is it the supernatant at the last step i TCA precipitate? if it is, i've looked at a protocol for TCA precipitation and if i followed that, i'd only be left with 2 components. spin down 2 ml cells at 4000 rpm, 4°C for 2 min 3. The datasets in XML format 0 20 40 60 80 100 120 420 440 460 480 500 520 540 560 580 600 620 Relative Intensity (%) Wavelength (nm) Native-Rnase Rnase-TCA Rnase-Ethanol/HCl Rnase-Ethanol Rnase-Acetone Fig. • After SDS-PAGE the proteins are visualized either by staining the gel or by western blotting. Jan 1, 2014 · Trichloroacetic acid (TCA) precipitation of proteins is commonly used to concentrate protein samples or remove contaminants, including salts and detergents, prior to downstream applications such as SDS-PAGE or 2D-gels. Therefore, aqueous TCA 60 precipitation is not commonly used like TCA/acetone precipitation in proteomic analysis. Apr 1, 2018 · Proteins extraction was performed on 500 mg of soil, 10 mg of biofilm and 15 mg of mouse liver as starting material according to protocols of Chourey et al. Protein precipitation by TCA precipitation to remove excess click reagents (steps 19–23). A number of methods are available for concentrating dilute protein solutions and simultaneously removing contaminating substances. Centrifuge at 3,000g for 30 min at 4 Modified TCA/Acetone precipitation of proteins The modified protocol is designed for running in Eppendorf tubes within 1 h and can be reli-ably adapted to big volumes. 67 mL of 1. • A single precipitation may not be sufficient to remove all types and concentrations of interfering contaminants. The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. 0 10% Trichloroacetic acid (TCA) 25 mM NH4OAc 1 mM Na2EDTA. Pool the 4 tubes of protein solution into a Falcon 50mL. 2 c). • TCA: 55% (w/v) trichloroacetic acid in H2O (stored at 4 deg) • HU buffer: 200 mM phosphate buffer, pH 6. Incubate on ice for at least 1 hr. Suspend lysed or disrupted sample in 10% TCA in acetone with either 0. I am not sure the low efficiency is caused by the high concentration of the sucrose, b/c I have the same issue with my system. • Ethanol/HCl represents a quick and inexpensive purification agent for proteomics studies. TCA precipitation denatures the protein, so it should not be used if the protein must remain in its folded state (e. Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample. add 100 μl of 10% TCA to 100 μl of protein lysate). 1 (Cleaning up samples using 2-D Clean-Up Kit) describes the kit and provides a protocol for use. TCA protein precipitation Introduction The protocol of protein precipitation to concentrate the solution. 1). The use of TCA combinedwith DOC allows for precipitation of proteins down to the 3 µg level. Incubate on ice for at least 30 min. People say that the major problem with TCA precipitation is to get the precipitate to dissolve. Centrifuge for 15 min in a microcentrifuge at 4°C at maximum speed. Andrew J. TCA-induced protein precipitation curves The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. I add 25% of my sample vol. Organic solvents and TCA are particularly useful for the analysis of samples by SDS-PAGE followed by Western blotting as trichloroacetic acid (TCA). Spin at max for 15 minutes. Suck off all the sup with a drawn out pasteur pipette. According to our results, we therefore propose that protein enrichment should be performed by TCA-DOC precipitation method after 48 h at 95% of confluence in a serum-deprived culture medium. Let cells grow for 7 days 2. Jan 1, 2019 · 7. (for details, check Maniatias under TCA ppt ) Precipitation Protocol: 1. May 9, 2007 · Then to concentrate the IP'd proteins, I am trying TCA precipitation. Each kit can process 50 samples of up to 100 µL each. This method is useful for obtaining prote Nov 7, 2023 · This can be achieved using trichloroacetic acid (TCA) precipitation and acetone-based protocols [14, 15]. Sep 4, 2014 · By Klaus D. Emission spectra of RNASE (λ exc ¼ 400nm) at different Aug 1, 2018 · Compared to classical TCA/acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. HCl, pH 8. Dec 19, 2018 · Compared to classical TCA/ acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. Diluted samples may be left overnight. e. TCA precipitation denatures the protein, so it should not be used if TCA Precipitation (Supernatant) 1. 4. TCA is Comparison of urinary sample preparation approaches. TCA (trichloroacetic acid) precipitation is considered the most efficient protocol for precipitating proteins from dilute solution. 1) Make a 100% w/v TCA stock and store the solution at 4°C in a brown bottle. The protocol involves mixing cell lysate with TCA and acetone, precipitating proteins at -20°C, washing the precipitate with acetone to remove contaminants, and dissolving the protein pellet in rehydration buffer for downstream analysis such as 2D gel Add 100% (w/v) TCA (trichloroacetic acid, to the sample to bring the TCA concentration to 20%. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is pre-sented. Protocol for 5' End Labeling RNA › Protocol for Synthesizing Biotinylated aRNA › Protocol for Trichloroacetic Acid (TCA) Precipitation › Ribominus Transcriptome Isolation Kit (Yeast and Bacteria) › RNAqueous™-96 RNA Isolation Kit Automation Protocol › RNA Purification Protocols TCA/Acetone Protein Precipitation Protocol The following procedure is used to precipitate proteins from freshly prepared cell lysates containing some contaminants, such as salt, detergents, nucleic acids, and lipids that can interfere with the subsequent 2D analysis. Protocol Trichloroacetic Acid (TCA) Precipitation of Proteins . However, most of the Deoxycholate+TCA precipitation protocols seem to So is it the supernatant at the last step i TCA precipitate? if it is, i've looked at a protocol for TCA precipitation and if i followed that, i'd only be left with 2 components. In our hands, TCA/acetone precipitation, a very common method reported in the literature 24 and the basis for the protocols provided to us, did not reliably generate reproducible pellets that could be resolubilized to obtain total protein concentrations with low variability. The precipitation of protein by TCA is specifically due to two properties, the acidity of TCA and the trichloro moiety that bear that molecule [16,17]. Proteins were precipitated with 25% (w/v) trichloroacetic acid (TCA). Carefully remove all supernatant. Alternatively, tissue may be homogenized directly into 10–20% TCA (35, 47). Protein solutions often contain buffers, salts, or other Most protocols for TCA precipitation seem to recommend keeping solutions cold and placing the reaction on ice for ~30 min. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. If sample preparation requires precipitation, typically only one precipitation technique is employed. [4] respectively. caltech. the TCA/acetone precipitation and phenol extraction methods. TCA Buffer. However Sivaraman, et al J. We performed TCA precipitation (followed by washes with acetone). 5ml of 3. I found a precipitation protocol with Jul 1, 2017 · Alternative protocols have been proposed using acetone alone, methanol/chloroform , or a combination of pyrogallol red, molybdate, and methanol . Although we have not tested the limit of the minimum amount of starting materials required for the HILAQ, we believe lower amounts may be used. , add 250µl TCA to 1. vortex thoroughly 6. 5mg of total protein as starting material. Mar 16, 2007 · Rout Lab Protocol Last Modified by Anna Sophia McKenney 3/16/07 Whole Cell Lysis and TCA Precipitation (Rout Lab, 2006) This is the method for whole cell lysis and TCA precipitation, used by the Rout lab as of 1998-2005. This solution is best prepared by dilution of stock solutions. Learn how to precipitate proteins using trichloro acetic acid (TCA) for mass spectrometry analysis. 5ml tube with maximum vol. of aqueous TCA precipitation and TCA/acetone precipitation, we 62 here report a modified, rapid method of TCA/acetone precipitation of plant proteins for proteomic 63 analysis. For 15 min. Timing >8 h. Add 10 ml 20% TCA solution to the falcons with supernatant (20% w/v in H 2 O, CAUTION: TCA is a strong acid, wear gloves, googles and handle with care!) 8. , if you For precipitation before performing a BCA Protein Assay, see the Tech Tip #8: Eliminate interfering substances from samples for BCA Protein Assay. Vortex and let sit for 30min at 4C. 19. TCA precipitation leads to loss of most of the low molecular weight proteins (either due to TCA mediated degradation or during the acetone wash steps). TCA is a classic reagent for precipitating proteins;1 however, it is not effective in quantitatively precipitating proteins below the 30 µg level. Both UKS and R2D2 solutions contain high molarity of urea and must not be heated above 30°C (which may be tempting to solubilize urea), because isocyanate ions are produced that would result in protein carbamylation. Right now I am at step 2 (O/N). Carefully remove the supernatant 11. We tried resuspending the pellet in 4xSDS loading buffer, and we have the same issue - the protein will simply not dissolve. (TCA) and sodium deoxycholate (DOC) reagents. In such cases, repeated precipitation may be performed. If TCA precipitation does not work for your protein of interest, it is advisable to try variants of TCA precipitation (included in this protocol) or other methods of precipitation or concentration, such as acetone precipitation, salting out (see Salting out of proteins using ammonium sulfate precipitation), immunoprecipitation (you can extract Jul 23, 2018 · TCA precipitation. Stock Solutions: 100% (w/v) Trichloroacetic acid (TCA) recipe: dissolve 500g TCA (as shipped) into 350 ml dH 2 O, store at RT. Spin in microfuge at 4 deg. Precipitate overnight in -20°C. I usually use acetone + 0,1 HCl (for better precipitation) and maximal cooling, and the only way to dissolve precipitated protein with good result is to use urea (for example). However, the high speed I have attached the protocol for TCA precipitationa nd preparation of protein samples. It is especially effective for samples with a large proportion of protein, so alternate protocols Jan 1, 2014 · If TCA precipitation does not work for your protein of interest, it is advisable to try variants of TCA precipitation (included in this protocol) or other methods of precipitation or concentration, such as acetone precipitation, salting out (see Salting out of proteins using ammonium sulfate precipitation), immunoprecipitation (you can extract Data show a comprehensive evaluation of protein conformational changes throughout TCA precipitation and one single step purification with various solvents. 000 g, 4°C 10. For high protein concentrations the protein will precipitate immediately. of 2% DOC (deoxycholate). 0], 10 mL 100% TCA solution, 1. spin samples in microfuge at 15000 g, 4°C for 15 min A number of methods are available for concentrating dilute protein solutions and simultaneously removing contaminating substances. Add 3. Place a brown bottle of 100% acetone in the -20 ºC. These are noted at the end. repeat step 2 and 3 5. 0ml sample. Full Text. Always keep an acetone bottle at 4°C to prepare these solutions. Trichloroacetic acid (TCA) precipitation of proteins is commonly used to concentrate protein samples or remove contaminants, including salts and detergents, prior to downstream applications such as SDS-PAGE or 2D-gels. Spin at 4°C for 1 min, 17000 rpm. . Most protocols for TCA precipitation seem to recommend keeping solutions cold and placing the reaction on ice for ~30 min.