Sample Prep
Content
2D Electrophoresis Protein Sample Prep CyDye labeling
Every time you use the 2D equipment, you MUST sign the logbook. Keep all equipment/supplies keratin-free.
Contact: Debby Walthall or Hyuk-Kyu Seoh Rm: NSC 338/NSC 516 Tel: (404) 413-5363 or (404)413-5379;
Sample Prep
Sample Prep and IEF are absolutely critical for good results. Almost all problems with 2D can be traced back to these 2 steps especially Sample Prep. Before starting a project – DO YOUR HOMEWORK!! Read through these guides – between the GE manual and 3 users, there should be enough information to get you started. Try to find a paper or someone who has done 2D on your system. This could help considerably with sample prep. Be careful with contaminants in your sample/buffers – IEF is very sensitive to certain chemicals (see GE manual, p. 32, 33). Salt is the most common sample contaminant. The manual not only describes the contaminant, it gives the reason it can cause problems AND a method to remove the contaminant. The 2D Cleanup kit and 2D Quant kit work well and are HIGHLY recommended. Your first reaction to using the GE 2D Cleanup kit and 2D Quant kit is that it is too expensive. But in the long run, it will save you considerable time and money. There is also a GE Mini Dialysis kit for desalting your sample. You can get a crude concentration using a spec. or use the BCA Protein assay kit (Pierce, cat. #: 23227) or a Bradford assay. All proteins must be completely soluble under electrophoresis conditions - 1st dimension and 2nd dimension buffers. Different
treatments and conditions depend on your specific sample and vary widely. The effectiveness of solubilization depends on the choice of cell disruption method, protein concentration and dissolution method, choice of detergents and composition of sample solution. All of these need to be optimized for your specific system or separations may be incomplete or distorted. Notes: • Keep sample prep simple to avoid protein loss. It’s a trade-off you have to decide, additional sample prep may improve the quality of the final 2-D gel, but at the expense of some protein loss. • The cells should be disrupted so that there is minimal proteolysis. Cell disruption should be done at as low a temp. as possible and with minimal heat generation. Cell disruption ideally should be done directly into a strongly denaturing solution containing protease inhibitors. • Sample prep solutions should be freshly prepared or stored as frozen aliquots. Use high purity urea containing protease inhibitors. • Prepare the sample just prior to IEF or store samples in aliquots at –80oC. Do not thaw multiple times. • Proteins should be kept cold at all times. Do not let sit at RT. • Remove solid material by centrifugation. Solid particles and lipids will block the gel pores. • Do not heat proteins after adding urea. If the soln. is heated over 30oC, the protein might be modified. Read the Amersham manual for details about sample prep (Important: explains Pros and Cons of each method) • Different methods for cell disruption. • Different methods for protecting your samples from proteolysis. • Different Precipitation procedures • Removal of Contaminants – includes a table with different contaminants, why they should be removed and recommended methods for removal • What needs to go into the sample solution and why • Protein Quantitating
Key: LB SB RB EB
Lysis Buffer 2X Sample Buffer Rehydration Buffer Equilibration Buffer
Sample Prep 1. Following is a basic sample prep for 2D gels. Using CyDyes has additional requirements – see below. 2. Remove cell pellets and lysis buffer (LB) (see Buffers and Solutions below) from -70C. Place pellets in -20C and allow LB to thaw at room temp. 3. Vortex LB thoroughly. Making sure all precipitate dissolves. 4. Resuspend the cell pellet in Lysis buffer. Add 1 ml Lysis buffer/100 ml o/n. Gently rotate tube in your hand. Use a clean spatula to scrape pellet away from side of tube. Chop the pellet us as much as you can. No vortexing. You do not want any frothing now or when you sonicate the sample. Frothing causes proteins to aggregate. 5. Let sit for 20 min. in Lysis buffer on ice. 6. Sonication. a. Setup a beaker with ice water with a stir bar in the bottom and placed on a stir plate. Setup a clamp to hold the tube in place. b. Clean sonication tip with 3 rounds of ethanol and water. c. Following is the program used for Pseudomonas gram (-) Set total time for 3 min. Pulse 1 sec. on and 0.5 second off. d. Stop and swirl/push ice next to tube. As you are pulsing on and off, turn sonicator dial up slowly. Every 30 sec., stop and let sample sit on ice for 1 min. e. Repeat steps a-e for remaining samples. 7. Spin 10 min. at 12,000 x g. Remove supernatant. Should be amber color. Transfer lysate to microfuge tube and place on ice. 8. Remove 100 ul for 2D Cleanup kit. Do a 2D Quant (or other quantification method) before 2D Cleanup to make sure you don’t have too much protein. You don’t want more than 100 ug protein/tube. If there is too much protein in the 2D Quant, the pellet wil be too large to resuspend effectively. Base
Cleanup volume on protein concentration not volume. Do multiple duplicates of your samples to increase total concentration and volume. 9. Store remaining protein -80oC. CyDyes Notes • CyDyes are VERY sensitive to pH. After 2D cleanup, resuspend sample in 30 mM Tris, pH 8.8 (pH needs to be between 8.0 and 9.0). • ABI and Roberto recommendation: If you are doing a big experiment, you need to randomize your samples and do dye swapping (Don’t label all controls with Cy3 and all samples with Cy5 – mix them up). This helps prevent any Dye bias and aids statistical analysis. • Running repetitive gels that swap the dyes used to label samples will control for any dye-specific effects that might result from preferential labeling or different fluorescence characteristics of the gel or glass plates at the different excitation wavelengths of Cy2, Cy3, and Cy5. For example, one set of gels might use Cy3 to label controls and Cy5 to label drug-treated samples. A duplicate set of gels should be run that uses Cy5 to label controls and Cy3 to label drug-treated samples. • You also need to do at least 3 biological replicates, not replicates of the same sample. • Control and sample variant depend on type of experiment. For example, control could be uninduced and variant could be induced; or control could be a 0 time point and variant could be 5min/10 min/15 min. etc. time point. Whatever your experiment is - you are comparing 2 conditions on 1 gel. You would need enough control protein to run with however many variants you have. For example, if you have 4 time points you would run 4 gels with each gel having a 0 time point as control and 1 time point as variant. Reasons to use all 3 CyDyes • Highly recommend using Cy2 as an internal standard. This is created by pooling an aliquot of all biological samples in the experiment. With the internal standard there is accurate quantification and accurate spot statistics between gels. To
•
•
•
fully utilize Decyder analysis software, an internal standard should be run with all gels. The software can determine protein abundance for each spot relative to the internal standard. Since all samples are compared to the same internal standard, cross-sample comparisons can be made, within the same gel or between multiple gels. Using the internal standard + Decyder software can detect and quantify differences as small as 10% between samples with greater than 95% confidence. You can run fewer gels using all 3 CyDyes.
Working Cy Dye solution (400pm/ul – Rollin; Anupama 200 pm/ul): 1. Terms. a. Dry CyDye stock from GE; no DMF added yet. Store at –20oC until expiration date. b. Dye stock solution. DMF added (see step 4). 1 mM stock stored at –20oC for up to 2 months or up to expiration date. c. Working dye solution (see step 5). 200 (step 7) - 400 (step 6) pmole stock stored at –20oC for only 2 weeks. 2. Remove dry CyDyes (10 nmol) from –20oC. 3. Transfer small amount of fresh DMF to a tube labeled DMF 4. See Supply list. Once DMF has been opened, it has a 3 month shelf life. I found a 25 ml DMF bottle from from Aldrich. 5. Dye Stock solution. Reconstitute CyDye (10nmol) in 10ul of fresh DMF (dimethyl formamide). This gives a stock solution of 1 mM. Vortex for 30 sec. Spin down. Immediately store at 20C. 6. Table for making Working dye solution – 400 pM: a. to tubes labeled cy2,3, and 5 add DMF (prior to dye): 4 samples __add 3ul DMF to tube 6 samples __ 4.5ul DMF to tube 8 samples __ 5.25ul DMF to tube b. Add the Dye stock solution: 4 samples __add 2ul Dye stock solution 6 samples __add 3ul Dye stock solution 8 samples __add 3.5ul Dye stock solution c. return Dye stock solution to -20C immediately
d. vortex ea Working dye solution for 10 sec and spin briefly e. USE IMMEDIATELY OR STORE AT -20oC 7. Table for making Working dye solution – 200 pM: a. to tubes labeled cy2,3, and 5 add DMF (prior to dye): 4 samples __add 3.2ul DMF to tube b. Now add the Dye stock solution 4 samples __add 0.8ul Dye stock solution 8. return Dye stock solution to -20C immediately 9. vortex ea for 10 sec and spin briefly 10. USE IMMEDIATELY OR STORE AT -20oC Sample CyDye labeling 1. Prepare protein sample as above. a. No 2D cleanup kit. After sonication and centrifugation, check to make sure the pH is between 8.0 – 9.0. See step 2 if you need to adjust the pH. b. 2D cleanup kit. At the end of the cleanup kit protocol, resuspend the pellet in 30 mM Tris, pH 8.8. 2. To adjust the pH of your sample. a. pH is too low. Make an identical cell lysis buffer without the protein at pH 9.5. Mix increasing volumes of the new lysis buffer with the protein sample. This will increase the pH, stop when the pH is 8.5. b. pH is too high. Make an identical cell lysis buffer without the protein at pH 7.5. Mix increasing volumes of the new lysis buffer with the protein sample. This will lower the pH, stop when the pH is 8.5. c. Alternatively, the pH can be increased to pH 8.5 by the careful addition of 50 mM NaOH. 3. Remove reconstituted dyes from –20oC and allow to thaw on ice for 5 min 4. after thawing, vortex ea dye and spin down briefly. Rollin. Dilute ea sample to 5mg/ml with LB to a final volume of 100 ul. Divide the [sample] in ug/ul into 375ug (75 ul of a 5 ug/ul solution)to determine appropriate sample volume. For example, sample concentration is 7.5 mg/ml or ug/ul, divide 375/7.5 = 75 ul sample + 25ul LB to get a final volume of 100 ul. This is enough
sample for analyticals, several preparative gels and a small amount to archive. Anupama. Need 50 ug/sample. Divide the [sample] in ug/ul into 50 ug to determine appropriate sample volume. For example, sample concentration is 7.5 mg/ml or ug/ul, divide 50/7.5 = 6.67 ul sample. Table for Dye Swapping. 12 samples
Gel Cy2 Standard Cy3 Cy5
1 2 3 4 5 6
50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4
of of of of of of
50ug Sample A1 50ug Sample B1 50ug Sample C1 50ug Sample A2 50ug Sample B2 50ug Sample C2
50 ug Sample C3 50 ug Sample A3 50 ug Sample B3 50 ug Sample C4 50 ug Sample A4 50 ug Sample B4
Besides switching dyes between samples, run replicates on different gels. Reaction Prep. CyDyes using Cy3, Cy5 and Cy2 1. Thaw CyDye stock solution and dilute or thaw diluted Working Dye solution. Keep on ice while thawing. 2. Label tubes. a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70ul) c. 1A cy3 d. 2A cy3 e. 3A cy3 f. 4B cy3 g. 5B cy3 h. 6B cy3 i. 1B cy5 j. 2B cy5 k. 3B cy5 l. 4A cy5
m. 5A cy5 n. 6A cy5 OR (same control for all gels a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70ul) c. control cy3 (3 gels + 0.5 = 3.5; 3.5 x 10u l = 35ul) d. 4B cy3 10 e. 5B cy3 f. 6B cy3 g. control cy5 (3 gels + 0.5 = 3.5; 3.5 x 10ul = 35ul) h. 1B cy5 i. 2B cy5 j. 3B cy5 3. Transfer 55 ul of each diluted sample/control to single tube labeled pool. Vortex pooled sample and place on ice. 4. Multiply number of analytical gels plus one by 10ul (6 gels + 1 = 7; 7 x 10ul = 70ul). Transfer this amount of pooled sample to tube labeled standard. This will be enough standard for each analytical gel and a little left over. Vortex standard sample and place on ice. The remaining pooled sample will to used for preparative loads and archiving. 5. Transfer 10 ul of ea diluted sample to corresponding reaction tube. 6. Transfer 10 ul of ea diluted control to corresponding reaction tube or if same control for all gels - 10 ul per # of gels into reaction tube labeled control. 7. Vortex and spin thawed working CyDye solution briefly. 8. Add dyes to appropriate tubes. Different control for each gel: a. 1A: 1ul cy3 b. 2A: 1ul cy3 c. 3A: 1ul cy3 d. 4B: 1ul cy3 e. 5B: 1ul cy3 f. 6B: 1ul cy3 g. 1B: 1ul cy5 h. 2B: 1ul cy5 i. 3B: 11ul cy5 j. 4A: 1ul cy5
k. 5A: 1ul cy5 l. 6A: 1ul cy5 OR (same control for all gel a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70 ul) c. control cy3 1ul x # of gels = 3 ul d. 4B: 1ul cy3 10 e. 5B: 1ul cy3 f. 6B: 1ul cy3 g. control cy5 1ul x # of gels = 3 ul h. 1B: 1ul l cy5 i. 2B: 1ul cy5 j. 3B: 1ul cy5 9. Vortex (10 sec) before returning to ice. 10. add 1ul cy2 per # of gels or per 10ul standard to tube labeled standard. 11. spin samples briefly and incubate on ice for 25 min protected from light. 12. add 1ul 10mM lysine per ul dye added previously to ea sample. 13. vortex for 5 sec. Incubate for 10 min on ice protected from light. 14. store samples in labeled box at -70 or proceed to reduction and 1stD. 15. archive remaining pool sample for prep gels. Prep for 1st Dimension. 1. Remove aliquot of 2X SB/RB buffer from - 70 if frozen. Once thawed, vortex well. Prepare 2X Sample Buffer (2XSB) and Rehydration buffer with or without Destreak. 2. CyDye samples a. Remove CyDye labeled samples, and pooled sample from -70 if frozen. Once thawed, vortex each. Ensure precipitates are dissolved. Spin down labeled samples. b. For each sample set (sample + control), combine Cy3 and Cy5 samples by transferring entire contents of Cy5 tubes to Cy3 tubes. c. Add 12ul standard to each Cy3 tube. Now each Cy3 tube should contain 36ul (10 ul control + 10 ul sample + 12 ul standard. d. Add 36ul 2XSB to each sample and vortex.
e. Transfer amount of pooled sample needed for preparative gels to fresh tube. Add equal volume of 2XSB and vortex. f. Incubate samples on ice in the dark for 30 min. 3. Non CyDye Samples a. Combine appropriate amount of sample (see Starting Decisions to be made for appropriate amount of protein for your detection method) with an equal volume of 2X SB. You want 1/8 – 1/4 of the 1st Dimension mix to be your sample + SB. Most of your volume will be RB. b. Incubate on ice for 30 min. c. Add appropriate volume of RB – depends on what size boats you are using. Buffers and Solutions Read the Amersham manual for basic solutions • Appendix 1 has solutions and buffers used to run the gels. Lysis buffer 30mM Tris, 7M urea, 2M thiourea, 4% CHAPS 13.2 ml deionized urea/thiourea solution 450 ul 1M Tris, not pH’d 150 ul GE nuclease mix 300 ul GE protease inhibitor 600 mg CHAPS pH to 8.6 with HCL Bring volume up to 15ml Aliquot 950 ul/tube and store at -70oC The 1X Rehydration Solution and 2X sample buffer both contain urea, thiourea (if present) and CHAPS. A stock can be prepared of these 2 (3) components, aliquoted and stored at –20oC. Immediately prior to use, DTT, Pharmalytes or IPG buffer are added to give either 2 X sample buffer or rehydration buffer. Once DTT, Pharmalyte or IPG buffer have been added, the solution is unstable and must be used the same day. All unused solution must be discarded. Buffer range is specific for the pI of the strip used and must be ordered separately.
Solution labeled 2X sample/Rehydration solution #1 or #2. It is actually 2X sample/1X rehydration solution. Rehydration buffer is NOT 2X. 2 X Sample/Rehydration 8M Urea, 4% CHAPS 20.2 ml 8 M Urea 1 gm CHAPS 4.8 ml dH2O Aliquot above (2.5 ml) and store for up to 6 months at –20oC. ** I had trouble getting the urea dissolved when I used dry urea. 2 X Sample/Rehydration solution 7M Urea, 2 M thiourea, 4% CHAPS #2: solution #1:
10.5 gm Urea 3.8 gm thiourea 1 gm CHAPS bring up to 25 ml with dH2O Aliquot (2.5 ml) and store for up to 6 months at –20oC. Rehydration Buffer (using DTT) 2X sample/rehydration solution (use buffer stock 1 or 2, depending on the rehydration buffer required), 1% Pharmalyte or IPG buffer, 0.2% (or 2 mg/ml or 13 mM) DTT 2.5 ml 2X sample/rehydration solution (1 or 2) 25 ul Pharmalyte or IPG buffer 5 mg DTT touch of Bromophenol Blue (dip yellow pipet tip into dye bottle and then into tube) Rehydration Buffer (using Destreak solution) 2 choices: 1. Destreak solution contains all of the components found in 2X sample buffer/rehydration buffer, just add appropriate IPG buffer 2. Destreak reagent contains only the GE proprietary reducing
agent. Combine with 2X sample buffer/rehydration buffer and appropriate IPG buffer. Put in frig the night before. Thaw on ice until ALL granules are dissolved. Vortex if needed. Add 0.5% IPG buffer to whole Destreak bottle solution - 15ul IPG buffer to 3ml, 60ul IPG buffer to 6ml. Whatever you don’t use – label bottle with date and whatever pH IPG buffer added. Store 20oC. 2X sample buffer 2X sample/rehydration solution (use buffer stock 1 or 2, depending on the rehydration buffer required), 2% Pharmalyte or IPG buffer, 2% (or 20 mg/ml or 130 mM) DTT 2.5 ml 2X sample/rehydration solution (1 or 2) 50 ul Pharmalyte or IPG buffer 50 mg DTT 40% CHAPS 20 gm CHAPS Make up to 50 ml with dH2O. Store at –20oC. Stable for 6 months. Urea/Thiourea solution 8M urea*, 2.3M thiourea 12 gm urea 4.5 gm thiourea Bring volume up to 25 ml with ddH2O Add 250 mg amberlite and stir for 1 h Filter into clean, rinsed bottle. *8M urea is almost at saturation.
Every time you use the 2D equipment, you MUST sign the logbook. Keep all equipment/supplies keratin-free.
Contact: Debby Walthall or Hyuk-Kyu Seoh Rm: NSC 338/NSC 516 Tel: (404) 413-5363 or (404)413-5379;
Sample Prep
Sample Prep and IEF are absolutely critical for good results. Almost all problems with 2D can be traced back to these 2 steps especially Sample Prep. Before starting a project – DO YOUR HOMEWORK!! Read through these guides – between the GE manual and 3 users, there should be enough information to get you started. Try to find a paper or someone who has done 2D on your system. This could help considerably with sample prep. Be careful with contaminants in your sample/buffers – IEF is very sensitive to certain chemicals (see GE manual, p. 32, 33). Salt is the most common sample contaminant. The manual not only describes the contaminant, it gives the reason it can cause problems AND a method to remove the contaminant. The 2D Cleanup kit and 2D Quant kit work well and are HIGHLY recommended. Your first reaction to using the GE 2D Cleanup kit and 2D Quant kit is that it is too expensive. But in the long run, it will save you considerable time and money. There is also a GE Mini Dialysis kit for desalting your sample. You can get a crude concentration using a spec. or use the BCA Protein assay kit (Pierce, cat. #: 23227) or a Bradford assay. All proteins must be completely soluble under electrophoresis conditions - 1st dimension and 2nd dimension buffers. Different
treatments and conditions depend on your specific sample and vary widely. The effectiveness of solubilization depends on the choice of cell disruption method, protein concentration and dissolution method, choice of detergents and composition of sample solution. All of these need to be optimized for your specific system or separations may be incomplete or distorted. Notes: • Keep sample prep simple to avoid protein loss. It’s a trade-off you have to decide, additional sample prep may improve the quality of the final 2-D gel, but at the expense of some protein loss. • The cells should be disrupted so that there is minimal proteolysis. Cell disruption should be done at as low a temp. as possible and with minimal heat generation. Cell disruption ideally should be done directly into a strongly denaturing solution containing protease inhibitors. • Sample prep solutions should be freshly prepared or stored as frozen aliquots. Use high purity urea containing protease inhibitors. • Prepare the sample just prior to IEF or store samples in aliquots at –80oC. Do not thaw multiple times. • Proteins should be kept cold at all times. Do not let sit at RT. • Remove solid material by centrifugation. Solid particles and lipids will block the gel pores. • Do not heat proteins after adding urea. If the soln. is heated over 30oC, the protein might be modified. Read the Amersham manual for details about sample prep (Important: explains Pros and Cons of each method) • Different methods for cell disruption. • Different methods for protecting your samples from proteolysis. • Different Precipitation procedures • Removal of Contaminants – includes a table with different contaminants, why they should be removed and recommended methods for removal • What needs to go into the sample solution and why • Protein Quantitating
Key: LB SB RB EB
Lysis Buffer 2X Sample Buffer Rehydration Buffer Equilibration Buffer
Sample Prep 1. Following is a basic sample prep for 2D gels. Using CyDyes has additional requirements – see below. 2. Remove cell pellets and lysis buffer (LB) (see Buffers and Solutions below) from -70C. Place pellets in -20C and allow LB to thaw at room temp. 3. Vortex LB thoroughly. Making sure all precipitate dissolves. 4. Resuspend the cell pellet in Lysis buffer. Add 1 ml Lysis buffer/100 ml o/n. Gently rotate tube in your hand. Use a clean spatula to scrape pellet away from side of tube. Chop the pellet us as much as you can. No vortexing. You do not want any frothing now or when you sonicate the sample. Frothing causes proteins to aggregate. 5. Let sit for 20 min. in Lysis buffer on ice. 6. Sonication. a. Setup a beaker with ice water with a stir bar in the bottom and placed on a stir plate. Setup a clamp to hold the tube in place. b. Clean sonication tip with 3 rounds of ethanol and water. c. Following is the program used for Pseudomonas gram (-) Set total time for 3 min. Pulse 1 sec. on and 0.5 second off. d. Stop and swirl/push ice next to tube. As you are pulsing on and off, turn sonicator dial up slowly. Every 30 sec., stop and let sample sit on ice for 1 min. e. Repeat steps a-e for remaining samples. 7. Spin 10 min. at 12,000 x g. Remove supernatant. Should be amber color. Transfer lysate to microfuge tube and place on ice. 8. Remove 100 ul for 2D Cleanup kit. Do a 2D Quant (or other quantification method) before 2D Cleanup to make sure you don’t have too much protein. You don’t want more than 100 ug protein/tube. If there is too much protein in the 2D Quant, the pellet wil be too large to resuspend effectively. Base
Cleanup volume on protein concentration not volume. Do multiple duplicates of your samples to increase total concentration and volume. 9. Store remaining protein -80oC. CyDyes Notes • CyDyes are VERY sensitive to pH. After 2D cleanup, resuspend sample in 30 mM Tris, pH 8.8 (pH needs to be between 8.0 and 9.0). • ABI and Roberto recommendation: If you are doing a big experiment, you need to randomize your samples and do dye swapping (Don’t label all controls with Cy3 and all samples with Cy5 – mix them up). This helps prevent any Dye bias and aids statistical analysis. • Running repetitive gels that swap the dyes used to label samples will control for any dye-specific effects that might result from preferential labeling or different fluorescence characteristics of the gel or glass plates at the different excitation wavelengths of Cy2, Cy3, and Cy5. For example, one set of gels might use Cy3 to label controls and Cy5 to label drug-treated samples. A duplicate set of gels should be run that uses Cy5 to label controls and Cy3 to label drug-treated samples. • You also need to do at least 3 biological replicates, not replicates of the same sample. • Control and sample variant depend on type of experiment. For example, control could be uninduced and variant could be induced; or control could be a 0 time point and variant could be 5min/10 min/15 min. etc. time point. Whatever your experiment is - you are comparing 2 conditions on 1 gel. You would need enough control protein to run with however many variants you have. For example, if you have 4 time points you would run 4 gels with each gel having a 0 time point as control and 1 time point as variant. Reasons to use all 3 CyDyes • Highly recommend using Cy2 as an internal standard. This is created by pooling an aliquot of all biological samples in the experiment. With the internal standard there is accurate quantification and accurate spot statistics between gels. To
•
•
•
fully utilize Decyder analysis software, an internal standard should be run with all gels. The software can determine protein abundance for each spot relative to the internal standard. Since all samples are compared to the same internal standard, cross-sample comparisons can be made, within the same gel or between multiple gels. Using the internal standard + Decyder software can detect and quantify differences as small as 10% between samples with greater than 95% confidence. You can run fewer gels using all 3 CyDyes.
Working Cy Dye solution (400pm/ul – Rollin; Anupama 200 pm/ul): 1. Terms. a. Dry CyDye stock from GE; no DMF added yet. Store at –20oC until expiration date. b. Dye stock solution. DMF added (see step 4). 1 mM stock stored at –20oC for up to 2 months or up to expiration date. c. Working dye solution (see step 5). 200 (step 7) - 400 (step 6) pmole stock stored at –20oC for only 2 weeks. 2. Remove dry CyDyes (10 nmol) from –20oC. 3. Transfer small amount of fresh DMF to a tube labeled DMF 4. See Supply list. Once DMF has been opened, it has a 3 month shelf life. I found a 25 ml DMF bottle from from Aldrich. 5. Dye Stock solution. Reconstitute CyDye (10nmol) in 10ul of fresh DMF (dimethyl formamide). This gives a stock solution of 1 mM. Vortex for 30 sec. Spin down. Immediately store at 20C. 6. Table for making Working dye solution – 400 pM: a. to tubes labeled cy2,3, and 5 add DMF (prior to dye): 4 samples __add 3ul DMF to tube 6 samples __ 4.5ul DMF to tube 8 samples __ 5.25ul DMF to tube b. Add the Dye stock solution: 4 samples __add 2ul Dye stock solution 6 samples __add 3ul Dye stock solution 8 samples __add 3.5ul Dye stock solution c. return Dye stock solution to -20C immediately
d. vortex ea Working dye solution for 10 sec and spin briefly e. USE IMMEDIATELY OR STORE AT -20oC 7. Table for making Working dye solution – 200 pM: a. to tubes labeled cy2,3, and 5 add DMF (prior to dye): 4 samples __add 3.2ul DMF to tube b. Now add the Dye stock solution 4 samples __add 0.8ul Dye stock solution 8. return Dye stock solution to -20C immediately 9. vortex ea for 10 sec and spin briefly 10. USE IMMEDIATELY OR STORE AT -20oC Sample CyDye labeling 1. Prepare protein sample as above. a. No 2D cleanup kit. After sonication and centrifugation, check to make sure the pH is between 8.0 – 9.0. See step 2 if you need to adjust the pH. b. 2D cleanup kit. At the end of the cleanup kit protocol, resuspend the pellet in 30 mM Tris, pH 8.8. 2. To adjust the pH of your sample. a. pH is too low. Make an identical cell lysis buffer without the protein at pH 9.5. Mix increasing volumes of the new lysis buffer with the protein sample. This will increase the pH, stop when the pH is 8.5. b. pH is too high. Make an identical cell lysis buffer without the protein at pH 7.5. Mix increasing volumes of the new lysis buffer with the protein sample. This will lower the pH, stop when the pH is 8.5. c. Alternatively, the pH can be increased to pH 8.5 by the careful addition of 50 mM NaOH. 3. Remove reconstituted dyes from –20oC and allow to thaw on ice for 5 min 4. after thawing, vortex ea dye and spin down briefly. Rollin. Dilute ea sample to 5mg/ml with LB to a final volume of 100 ul. Divide the [sample] in ug/ul into 375ug (75 ul of a 5 ug/ul solution)to determine appropriate sample volume. For example, sample concentration is 7.5 mg/ml or ug/ul, divide 375/7.5 = 75 ul sample + 25ul LB to get a final volume of 100 ul. This is enough
sample for analyticals, several preparative gels and a small amount to archive. Anupama. Need 50 ug/sample. Divide the [sample] in ug/ul into 50 ug to determine appropriate sample volume. For example, sample concentration is 7.5 mg/ml or ug/ul, divide 50/7.5 = 6.67 ul sample. Table for Dye Swapping. 12 samples
Gel Cy2 Standard Cy3 Cy5
1 2 3 4 5 6
50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4 50 ug (4.17 ug each A1-4, B1-4, C 1-4
of of of of of of
50ug Sample A1 50ug Sample B1 50ug Sample C1 50ug Sample A2 50ug Sample B2 50ug Sample C2
50 ug Sample C3 50 ug Sample A3 50 ug Sample B3 50 ug Sample C4 50 ug Sample A4 50 ug Sample B4
Besides switching dyes between samples, run replicates on different gels. Reaction Prep. CyDyes using Cy3, Cy5 and Cy2 1. Thaw CyDye stock solution and dilute or thaw diluted Working Dye solution. Keep on ice while thawing. 2. Label tubes. a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70ul) c. 1A cy3 d. 2A cy3 e. 3A cy3 f. 4B cy3 g. 5B cy3 h. 6B cy3 i. 1B cy5 j. 2B cy5 k. 3B cy5 l. 4A cy5
m. 5A cy5 n. 6A cy5 OR (same control for all gels a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70ul) c. control cy3 (3 gels + 0.5 = 3.5; 3.5 x 10u l = 35ul) d. 4B cy3 10 e. 5B cy3 f. 6B cy3 g. control cy5 (3 gels + 0.5 = 3.5; 3.5 x 10ul = 35ul) h. 1B cy5 i. 2B cy5 j. 3B cy5 3. Transfer 55 ul of each diluted sample/control to single tube labeled pool. Vortex pooled sample and place on ice. 4. Multiply number of analytical gels plus one by 10ul (6 gels + 1 = 7; 7 x 10ul = 70ul). Transfer this amount of pooled sample to tube labeled standard. This will be enough standard for each analytical gel and a little left over. Vortex standard sample and place on ice. The remaining pooled sample will to used for preparative loads and archiving. 5. Transfer 10 ul of ea diluted sample to corresponding reaction tube. 6. Transfer 10 ul of ea diluted control to corresponding reaction tube or if same control for all gels - 10 ul per # of gels into reaction tube labeled control. 7. Vortex and spin thawed working CyDye solution briefly. 8. Add dyes to appropriate tubes. Different control for each gel: a. 1A: 1ul cy3 b. 2A: 1ul cy3 c. 3A: 1ul cy3 d. 4B: 1ul cy3 e. 5B: 1ul cy3 f. 6B: 1ul cy3 g. 1B: 1ul cy5 h. 2B: 1ul cy5 i. 3B: 11ul cy5 j. 4A: 1ul cy5
k. 5A: 1ul cy5 l. 6A: 1ul cy5 OR (same control for all gel a. Pool b. Standard (6 gels + 1 = 7; 7 x 10ul = 70 ul) c. control cy3 1ul x # of gels = 3 ul d. 4B: 1ul cy3 10 e. 5B: 1ul cy3 f. 6B: 1ul cy3 g. control cy5 1ul x # of gels = 3 ul h. 1B: 1ul l cy5 i. 2B: 1ul cy5 j. 3B: 1ul cy5 9. Vortex (10 sec) before returning to ice. 10. add 1ul cy2 per # of gels or per 10ul standard to tube labeled standard. 11. spin samples briefly and incubate on ice for 25 min protected from light. 12. add 1ul 10mM lysine per ul dye added previously to ea sample. 13. vortex for 5 sec. Incubate for 10 min on ice protected from light. 14. store samples in labeled box at -70 or proceed to reduction and 1stD. 15. archive remaining pool sample for prep gels. Prep for 1st Dimension. 1. Remove aliquot of 2X SB/RB buffer from - 70 if frozen. Once thawed, vortex well. Prepare 2X Sample Buffer (2XSB) and Rehydration buffer with or without Destreak. 2. CyDye samples a. Remove CyDye labeled samples, and pooled sample from -70 if frozen. Once thawed, vortex each. Ensure precipitates are dissolved. Spin down labeled samples. b. For each sample set (sample + control), combine Cy3 and Cy5 samples by transferring entire contents of Cy5 tubes to Cy3 tubes. c. Add 12ul standard to each Cy3 tube. Now each Cy3 tube should contain 36ul (10 ul control + 10 ul sample + 12 ul standard. d. Add 36ul 2XSB to each sample and vortex.
e. Transfer amount of pooled sample needed for preparative gels to fresh tube. Add equal volume of 2XSB and vortex. f. Incubate samples on ice in the dark for 30 min. 3. Non CyDye Samples a. Combine appropriate amount of sample (see Starting Decisions to be made for appropriate amount of protein for your detection method) with an equal volume of 2X SB. You want 1/8 – 1/4 of the 1st Dimension mix to be your sample + SB. Most of your volume will be RB. b. Incubate on ice for 30 min. c. Add appropriate volume of RB – depends on what size boats you are using. Buffers and Solutions Read the Amersham manual for basic solutions • Appendix 1 has solutions and buffers used to run the gels. Lysis buffer 30mM Tris, 7M urea, 2M thiourea, 4% CHAPS 13.2 ml deionized urea/thiourea solution 450 ul 1M Tris, not pH’d 150 ul GE nuclease mix 300 ul GE protease inhibitor 600 mg CHAPS pH to 8.6 with HCL Bring volume up to 15ml Aliquot 950 ul/tube and store at -70oC The 1X Rehydration Solution and 2X sample buffer both contain urea, thiourea (if present) and CHAPS. A stock can be prepared of these 2 (3) components, aliquoted and stored at –20oC. Immediately prior to use, DTT, Pharmalytes or IPG buffer are added to give either 2 X sample buffer or rehydration buffer. Once DTT, Pharmalyte or IPG buffer have been added, the solution is unstable and must be used the same day. All unused solution must be discarded. Buffer range is specific for the pI of the strip used and must be ordered separately.
Solution labeled 2X sample/Rehydration solution #1 or #2. It is actually 2X sample/1X rehydration solution. Rehydration buffer is NOT 2X. 2 X Sample/Rehydration 8M Urea, 4% CHAPS 20.2 ml 8 M Urea 1 gm CHAPS 4.8 ml dH2O Aliquot above (2.5 ml) and store for up to 6 months at –20oC. ** I had trouble getting the urea dissolved when I used dry urea. 2 X Sample/Rehydration solution 7M Urea, 2 M thiourea, 4% CHAPS #2: solution #1:
10.5 gm Urea 3.8 gm thiourea 1 gm CHAPS bring up to 25 ml with dH2O Aliquot (2.5 ml) and store for up to 6 months at –20oC. Rehydration Buffer (using DTT) 2X sample/rehydration solution (use buffer stock 1 or 2, depending on the rehydration buffer required), 1% Pharmalyte or IPG buffer, 0.2% (or 2 mg/ml or 13 mM) DTT 2.5 ml 2X sample/rehydration solution (1 or 2) 25 ul Pharmalyte or IPG buffer 5 mg DTT touch of Bromophenol Blue (dip yellow pipet tip into dye bottle and then into tube) Rehydration Buffer (using Destreak solution) 2 choices: 1. Destreak solution contains all of the components found in 2X sample buffer/rehydration buffer, just add appropriate IPG buffer 2. Destreak reagent contains only the GE proprietary reducing
agent. Combine with 2X sample buffer/rehydration buffer and appropriate IPG buffer. Put in frig the night before. Thaw on ice until ALL granules are dissolved. Vortex if needed. Add 0.5% IPG buffer to whole Destreak bottle solution - 15ul IPG buffer to 3ml, 60ul IPG buffer to 6ml. Whatever you don’t use – label bottle with date and whatever pH IPG buffer added. Store 20oC. 2X sample buffer 2X sample/rehydration solution (use buffer stock 1 or 2, depending on the rehydration buffer required), 2% Pharmalyte or IPG buffer, 2% (or 20 mg/ml or 130 mM) DTT 2.5 ml 2X sample/rehydration solution (1 or 2) 50 ul Pharmalyte or IPG buffer 50 mg DTT 40% CHAPS 20 gm CHAPS Make up to 50 ml with dH2O. Store at –20oC. Stable for 6 months. Urea/Thiourea solution 8M urea*, 2.3M thiourea 12 gm urea 4.5 gm thiourea Bring volume up to 25 ml with ddH2O Add 250 mg amberlite and stir for 1 h Filter into clean, rinsed bottle. *8M urea is almost at saturation.
