Protein Precipitation Protocols Notes: All reagents need to high purity/hplc quality. All tubes used should be new or hand cleaned thoroughly with Micro90 detergent. High quality water needs to be used for all the solutions and steps. For this protocol, I used terminology MilliQ water to mean that equivalent quality of water needs to be used. Therefore, never use dh 2 O or ddh 2 O water. At all steps of the sample preparation, use powder-free nitrile gloves (no aloe). When using precipitation as a cleanup step, it is recommended to have a starting protein concentration in the range of 1 10 mg/ml. When protein samples are too diluted, it will be difficult to quantitatively recover proteins following precipitation cleanup (Friedman, D.B., Hoving, S., and Westermeier, R. (2009) Isoelectric focusing and two-dimensional gel electrophoresis. Methods in Enzymology 463: 515-540). I find this may not be possible for many samples. Acetone Precipitation [Modified from the protocol posted by the Nevada Proteomics Center] http://www.unr.edu/inbre/proteomics/acetone_precipitation_protocol.html 1) Prepare 80% acetone solution and store at -20 ºC in a brown bottle. 2) Add 4 volumes chilled acetone/milliq water stock to the sample extract. 3) Vortex well. 4) Incubate tube overnight at -20 ºC. (The tubes can be left up to 1 week with minimal protein degradation or modification (GE 2-D Clean-Up Kit)). 5) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg, 6) Gently dump off the supernatant. 7) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly 8) Pipet off the remaining sup, making sure no visible liquid is left behind. 9) Wash the pellet with 1 ml (microfuge tubes) or 10 ml (larger tubes) chilled acetone/milliq. The pellet needs to be thoroughly broken up with a glass rod. 10) Repeat steps #5-8. 11) Repeat wash 1 more time (steps #9-10). 12) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend. 13) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.) TCA Precipitation with Acetone Wash 1) Make a 100% w/v TCA stock and store the solution at 4 C in a brown bottle. Place a brown bottle of 100% acetone in the -20 ºC. 2) To two samples, add chilled 100% TCA at1/10 of the sample volume. 3) Mix sample by inverting several times. 4) Incubate one of the samples on ice for 15 min and incubate the other overnight at -20 ºC. Overnight increases yields but could damage sialic acid on glycoproteins. 5) Thaw -20 ºC sample. 6) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg, 7) Gently dump off the supernatant. 8) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly 9) Pipet off the remaining sup, making sure no visible liquid is left behind. 10) Suspend the pellet in 100 μl (microfuge tube) and 1 ml (large centrifuge tube) milliq water using a glass rod. The pellet should be dispersed, but will not be dissolved by the water. 11) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) chilled 100% acetone. 1
12) Vortex each tube for 30 sec. 13) Over a course of 1 hr, vortex the tube three times for 30 sec. Continue incubating the tubes at -20 ºC between each vortexing. 14) Incubate tubes at -20 ºC overnight. (You can skip this step but your yields are lower. The tubes can be left up to 1 week with minimal protein degradation or modification (GE 2-D Clean-Up Kit)). 15) Repeat steps #6-9. 16) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend. 17) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.) TCA/Acetone Precipitation [Modified from the protocol described by Gorg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized ph gradients. Electrophoresis 21, 1037-53.] 1) Make a 13.3% w/v TCA in acetone stock and 100% acetone stock. Store the solutions in a brown bottle at -20 ºC. Add 0.2% w/v DTT to both solutions just prior to use. 2) Add three volumes of the chilled 13.3% w/v TCA in acetone containing 0.2% w/v DTT stock to the sample (final concentration is 10% TCA). Mix well by inverting tube. 3) Incubate for 1.5 hrs or overnight at -20 ºC. Overnight increases yields but could damage sialic acid on glycoproteins. 4) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg, 5) Dump off the supernatant. 6) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly 7) Pipet off the remaining sup, making sure no visible liquid is left behind. 8) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone containing 0.2% DTT stock. Break up the pellet using a glass rod. The pellet should be dispersed, but will not be dissolved by the water. 9) Vortex each tube for 30 sec. 10) Over a course of 1 hr, vortex the tube three times for 30 sec. 11) Repeat centrifugation steps #4-7. 12) Repeat the acetone Wash 1X (steps #8-11). 13) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend. 14) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.) Peterson TCA-DOC Precipitation with Acetone Wash [This method was recommended by Hediye Erdujument-Bromage from the Proteomics Core of the Memorial Sloan-Kettering Cancer Center for precipitating samples with low concentrations of proteins. TCA-DOC part of this protocol was slightly modified by us from the manuscript from Peterson, G. (1977) A Simplification of the Protein Assay Method of Lowry et al. Which is More Generally Applicable. Analytical Biochemistry 83: 346-356.] 1) Make a 72% TCA stock and store the solution at 4 ºC in a brown bottle. Place a brown bottle of 100% acetone in the -20 C. Make a 0.15% solution of sodium deoxycholate and store at room temperature. 2) To the sample, add 1/10 of the volume of 0.15% sodium deoxycholate. Mix by inverting the tube several times. 3) Incubate on ice for 15 min. 4) Add 1/10 of the original sample volume of the chilled 72% TCA. Mix by inverting the tube several times. 5) Incubate on ice for 15 min. 2
6) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg, 7) Dump off the supernatant. 8) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly 9) Pipet off the remaining sup, making sure no visible liquid is left behind. 10) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone. (The tubes can be left up to 1 week with minimal protein degradation or modification (GE 2-D Clean-Up Kit)). 11) Vortex each tube for 30 sec.` 12) Over a course of 1 hr, vortex the tube three times for 30 sec. 13) Repeat centrifugation steps #6-9. 14) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend. 15) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.) Arnold TCA-DOC Precipitation with Acetone Wash [This is method was modified from the original method published by Arnold and Ulbrich-Hofmann (Arnold, U. and Ulbrich-Hofmann, A. (1999) Quantitative Protein Precipitation from Guanidine Hydrochloride-Containing Solutions by Sodium Deoxycholate/Trichloroacetic Acid Analytical Biochemistry 271: 197-199). The original method was referenced by Friedman et al. ] 1) Make a 50% TCA stock and store the solution at 4 ºC in a brown bottle. Place a brown bottle of 100% acetone in the -20 C. Make a 1% solution of sodium deoxycholate and store at room temperature. 2) To the sample, add 1/10 of the volume of 1% sodium deoxycholate. Mix by inverting the tube several times. 3) Incubate on ice for 15 min. 4) Add 1/5 of the original sample volume of the chilled 50% TCA. Mix by inverting the tube several times. 5) Incubate on ice for 15 min. 6) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg, 7) Dump off the supernatant. 8) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly 9) Pipet off the remaining sup, making sure no visible liquid is left behind. 10) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone. (The tubes can be left up to 1 week with minimal protein degradation or modification). 11) Vortex each tube for 30 sec.` 12) Over a course of 1 hr, vortex the tube three times for 30 sec. 13) Repeat centrifugation steps #6-9. 14) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend. 15) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.) Methanol and Chloroform Precipitation [This method was adapted by David Friedman (Friedman, D.B., Hoving, S., and Westermeier, R. (2009) Isoelectric focusing and two-dimensional gel electrophoresis. Methods in Enzymology 463: 515-540) from the method described by Wessel and Flugge (Wessel, D., and Flugge, U. I. (1984). A method for the Quantitative Recovery of Protein in Dilute Solution in the Presence of Detergents and Lipids. Analytical Biochemistry 138: 141 143). 1) Bring up predetermined amount of protein extract to 100 ml with water. 2) Add 300 ml (3-volumes) water. 3) Add 400 ml (4-volumes) methanol. 3
4) Add 100 ml (1-volume) chloroform. 5) Vortex vigorously and centrifuge; the protein precipitate should appear at the interface. 6) Remove the water/meoh mix on top of the interface, being careful not to disturb the interface. Often the precipitated proteins do not make a visibly white interface, and care should be taken not to disturb the interface. 7) Add another 400 ml methanol to wash the precipitate. 8) Vortex vigorously and centrifuge; the protein precipitate should now pellet to the bottom of the tube. 9) Remove the supernatant and briefly dry the pellets in a vacuum centrifuge. 10) Resuspend the pellets in a suitable amount of 2D gel-compatible buffer Acidified Acetone and Methanol Precipitation [This method was posted by Kevin Hakala on the ABRF forum (3/6/13) in response on how to remove SDS- PAGE Sample Buffer from a protein sample. He noted the following: It recovers dilute protein from SDSsample buffer. I have not tried to digest the precipitated material, I only re-suspended the pellet in a smaller volume of SDS-sample buffer to concentrate the original sample. I don't have any data regarding recovery.] 1) Prepare acidified acetone: 120ml acetone + 10µl HCl (1mM final concentration). 2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20ºC. 3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep ON at -20ºC. 4) Spin 15min 4ºC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). 5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (small tubes). Resuspending the Pellets for Traditional SDS-PAGE Gel Electrophoresis 1) Store pellets at -80 ºC until ready to run the gel. 2) Resuspend the pellet by pipetting in 1X SDS sample buffer mixed with fresh reducing agent. 3) Briefly vortex the sample. (Skip this step if you are interested in studying protein modifications due to oxidization.) 4) Incubate sample at 70 C for 10 min or 95 C for 5 min. 5) Load sample onto the gel after cooling. Resuspending the Pellets for 2-D Gel Electrophoresis 1) Thaw the standard Rehydration Solution (7 M Urea + 2 M Thiourea + 4% CHAPS + 0.5% Ampholytes 3-10 + 0.0002% Bromophenol Blue). Add 0.004g DTT. 2) Add 300 μl of standard Rehydration Solution + DTT to each pellet. 3) Resuspend the pellets by pipetting. Don t vortex since it will oxidize the proteins. 4) Leave the sample resting on top of ice for 2 hrs, pipetting every 30 min to get the sample into solution. If your sample goes into solution sooner then proceed onto step #10. 5) If the sample has still not gone into solution, freeze the samples at -80 ºC to help the sample go into solution. 6) Thaw the samples at RT. 7) Resuspend the pellets by pipetting. If your sample goes into solution sooner then proceed onto step #10. 8) If the sample has still not go into solution repeat freeze thaw and resuspension steps (#5-7). If your sample goes into solution sooner then proceed onto step #10. 4
9) If the pellet still has not gone into solution, leave sample overnight at 4ºC. Resuspend the pellet the next morning by pipetting. 10) Transfer the samples to 1.5 ml microfuge tube. 11) Microfuge at top speed for 5 min at 4ºC. 12) Transfer the sup to another 1.5 ml microfuge tube. Make sure you pipet away from the pellet. 13) Take 30 μl of each sample to measure the protein conc. 14) Store the remaining samples and the 30 μl aliquots at -80 ºC. Note: If your pellet has not been solubilized after step #9, sonication maybe used. However, this should be used as a last resort since extreme care must be taken in not heating sample since urea breaks down during heating. The sample should be sonicated with bursts lasting no longer than several seconds, and should be chilled between bursts by placing on ice. 5
Measuring Protein Concentration of Cleaned-Up Samples with Modified Bradford Assay [Scaled down from the protocol described by Ramagli, L. S., Quantifying protein in 2-D PAGE solubilization buffers. Methods Mol Biol 1999, 112, 99-103.] 1) Thaw standard rehydration solution and add 0.004 g of DTT (0.4%) for every 1 ml rehydration solution. 2) Prepare fresh ovalbumin standards in Rehydration buffer (Standards can be stored at -20ºC and reused): For 10 mg/ml: Add 0.005g per 500 μl Rehydration buffer + DTT For 4 mg/ml: Add 200 μl 10mg/ml stock to 300 μl Rehydration buffer + DTT For 1 mg/ml: Add 50 μl 10mg/ml stock to 450 μl Rehydration buffer + DTT 3) Prepare Bio-Rad Protein Reagent by diluting 1:4 in milliq water. Diluted reagent does not need to be freshly made. 4) Add 20 μl 0.12 N HCl to all the standard and sample tubes. (Assuming concentration HCl is 12.1 N, add 10 ml conc HCl to 990 ml nanopure water to make a 0.12 N HCl) 5) Prepare protein standard curve (Do in duplicate): Final Conc. of Amt. of Ovalbumin (Std.) Rehyd. Sol n Std. Stock Used Amt. of Std. Stock 1) Blank 5 μl 0 μl 0 μl 2) 1 μg 4 μl 1 mg/ml 1 μl 3) 2 μg 3 μl 1 mg/ml 2 μl 4) 4 μg 1 μl 1 mg/ml 4 μl 5) 8 μg 3 μl 4 mg/ml 2 μl 6) 16 μg 1 μl 4 mg/ml 4 μl 7) 32 μg 1.8 μl 10 mg/ml 3.2 μl 6) Prepare the following samples (Do in quadruplicate): Sample Amt. of Amt. of Sample Rehyd. Sol n Sample 0 μl 5 μl Sample (if the blue color is higher than the standard curve) 4 μl 1 μl 7) Add 875 μl of Bio-Rad diluted dye to each tube. Immediately after addition, close the lid on the tube and mix by vortexing tube. 8) Incubate the tubes for 5 min at room temp. 9) Transfer 200 μl from each tube to a 96-well plate. Prior to transfer, mix the tube by vortexing. 10) Measue A 595 before 1 hr. 11) Use a linear curve to fit the standards. Melissa Sondej 7/6/11 6