Amersham™ CyDye DIGE Fluors - GE Healthcare Life Sciences
Contents
1. 6 2 Preparation of a cell lysate compatible with CyDye DIGE minimal labeling The example given here is that used with an Escherichia coli model system See Ettan DIGE user manual for more details Recommendations for different cell types are listed on page 24 Approximately 4 x 10 E coli cells will result in a 5 to 10 mg ml of protein in 1 ml of lysis buffer 12 1 Pellet the cells in a suitable centrifuge at 4 C 2 Pour off all growth media taking care not to disturb the cell pellet 3 Resuspend cell pellet in 1 ml of standard cell wash buffer in a microfuge tube 4 Pellet the cells in a microcentrifuge at 12 000 x g for 4 minutes at 4 C 5 Remove and discard the supernatant 6 Resuspend cell pellet in 1 ml of standard cell wash buffer in a microfuge tube 7 Repeat steps 4 to 6 at least three times 8 Ensure all the wash buffer has been removed with a fine pipette 9 Resuspend the washed cell pellet in 1 ml of lysis buffer 30 mM Tris 7 M Urea 2 M Thiourea 4 w v CHAPS pH 8 5 and leave on ice for 10 minutes Note if the protein concentration is less than 5 mg ml after protein quantitation resuspend cells in a correspondingly smaller volume of lysis buffer in subsequent experiments 10 Keep the cells on ice and sonicate intermittently until the cells are lysed See Cell sonication page 19 11 Centrifuge the cell lysate at 4 C for 10 minutes at 12 000 x gina microcentrifuge 12 Transfe2. Bulk labeling reactions can also be done using more protein and dye 2 Add 1 ul of diluted CyDye to the microfuge tube containing the protein sample i e 50 ug of protein is labeled with 400 pmol of dye for the labeling reaction 3 Mix and centrifuge briefly in a microcentrifuge Leave on ice for 30 minutes in the dark 4 Add 1 ul of 10 mM lysine to stop the reaction Mix by pipetting and spin briefly in a microcentrifuge 5 Leave for 10 minutes on ice in the dark 16 6 Samples can now be stored for at least three months at 70 C in the dark 6 6 Loading samples onto IPG strips 1 After the protein samples have been CyDye labeled add an equal volume of 2 x sample buffer and leave on ice for 10 minutes 2 Pool the protein samples that are going to be separated on the same 1 and 24 dimension gel There are now two options Option 1 The samples can be loaded onto a rehydrated IPG strip via cup loading on the Multiphor II or IPGphor Option 2 Follow the protocol outlined below if the IPG strips are to be rehydrated in the presence of the protein sample The total volume of labeled protein needs to be made up to the volume required for each IPG strip using the rehydration buffer One protein sample labeled with one CyDye 20 ul Add an equal volume 2 x sample buffer 20 ul 20 pl 40 pl Add three samples together 40 ul x 3 Total volume 120 ul A 24 cm IPG strip needs a total volume of 450 ul so add 45
3. 5 x 5 nmol of CyDye DIGE Fluor Cy5 minimal dye 25 8008 60 10 nmol 2 x 5 nmol of CyDye DIGE Fluor Cy2 minimal dye 25 8008 61 10 nmol 2 x 5 nmol of CyDye DIGE Fluor Cy3 minimal dye 25 8008 62 10 nmol 2 x 5 nmol of CyDye DIGE Fluor Cy5 minimal dye 25 8010 65 5 nmol of CyDye DIGE Fluor minimal labeling kit Cy2 Cy3 and Cy5 25 8010 82 5 nmol of CyDye DIGE Fluor Cy2 minimal dye 25 8010 83 5 nmol of CyDye DIGE Fluor Cy3 minimal dye 25 8010 85 5 nmol of CyDye DIGE Fluor Cy5 minimal dye 28 9345 30 2 nmol of CyDye DIGE Fluor minimal labeling kit Cy2 Cy3 and Cy5 4 Other materials required Reconstitution of CyDye 99 8 anhydrous Dimethylformamide DMF less than 3 months old from day of opening Sigma Aldrich 22 705 6 Labeling Microfuge tubes 1 5 ml Standard Cell wash buffer 10 mM Tris pH 8 0 5 mM Magnesium Acetate Store in aliquots at 15 C to 30 C Lysis buffer 30 mM Tris 7 M Urea 2 M Thiourea 4 w v CHAPS Adjust to pH 8 5 with dilute HCI Aliquots can be stored at 15 C to 30 C Lysine 10 mM L Lysine Sigma Aldrich L 5626 PH indicator strips Sigma pH test strips pH 4 5 10 0 P4536 For additional protocols 2 x Sample buffer 8 M Urea 130 mM DTT 4 w v CHAPS 2 v v Pharmalyte 3 10 for IEF Aliquots can be stored at 15 C to 30 C Rehydration buffer 8 M Urea 4 w v CHAPS 1 v v Pharmalyte 3 10 for IEF 13 mM DTT Sodium Hydroxide 50 mM NaOH 2 x Gel lo
4. disruption of dye required to label 50 pg protein Saccharomyces 2 M Thiourea Pellet washed with PBS 400 pmol cerevisiae 7 M Urea Cells sonicated on wetice 50 ug 30 mM Tris 4 and spun in a microfuge CHAPS pH to 8 5 for 5 min at 12 000 x g Pellet discarded and protein concentration determined Escherichia coli 7 M Urea Pellet sonicated on wetice 400 pmol 2 M Thiourea at amplitude 7 microns for 50 ug 30 mM Tris 4 30 seconds pulses Spun CHAPS pH to 8 5 in a centrifuge at 4 C at 12 000 x g for 10 minutes Pellet discarded and protein concentration determined Rat liver 7 M Urea 1 g of tissue placed in 10 mI 400 pmol 2 M Thiourea of lysis buffer Tissue then 50 pg 10 mM Tris homogenized and sonicated 5 mM Magnesium and lysate centrifuged at Acetate 10 C at 12 000 x g for 1 4 CHAPS pH hour Pellet then discarded to 8 0 and protein concentration determined Rat heart 7 M Urea g of tissue placed in 10 ml 400 pmol 2 M Thiourea of lysis buffer Tissue then 50 ug 10 mM TrispH8 homogenized and sonicated 5 mM Magnesium and lysate centrifuged at Acetate 10 C at 12 000 x g for 1 4 CHAPS pH hour Pellet then discarded to 8 5 and protein concentration determined Tomato fruit 7 M Urea Phenol based total protein 200 pmol wall 2 M Thiourea extraction including 50 ug 4 CHAPS homogenizing step 10 mM Tris pH to 8 5 For further examples of cell and tissue types tested with 2D DIGE please refer to the Ettan DIGE User Manual
5. doubt use an unopened batch of DMF for reconstituting the fluor 1 Take a small volume of DMF from its original container and dispense into a fresh microfuge tube 2 Remove the CyDye from the 15 C to 30 C freezer and leave to warm for 5 minutes at room temperature WG After 5 minutes add the specified volume of DMF to each new vial of CyDye see specification sheet supplied with the dye This gives a CyDye stock solution of 1 mM For the 2 nmol pack size this gives a CyDye working solution of 0 4 mM and should be used immediately 4 Replace the cap on the dye microfuge tube and vortex vigorously for 30 seconds 14 5 Centrifuge the microfuge tube for 30 seconds at 12 000 x g ina microcentrifuge 6 The fluor can now be used Unused CyDye stock solution should be returned to the 15 C to 30 C freezer as soon as possible and stored in the dark After reconstitution CyDye stock solution is only stable and usable until the expiry date detailed on the tube or for 2 months whichever is sooner 6 4 Preparation of CyDye working solution used to label proteins Note 2 nmol pack sizes do not need to undergo this process 1 ul of the 2 nmol CyDye working solution contains already 400 pmol 1 Briefly spin down CyDye stock solution prepared in protocol 3 in a microcentrifuge 2 Add one volume of CyDye stock solution to 1 5 volumes of high grade DMF to make 400 uM CyDye solution For example take 2 ul CyDye s
6. mM Tris without the protein at pH 9 5 2 Mix increasing volumes of the new lysis solution to the protein sample This will increase the pH of the protein sample as more lysis buffer is added Stop when the pH of the protein sample is at pH 8 5 Alternatively the pH of the lysate can be increased to pH 8 5 by the careful addition of dilute Sodium Hydroxide 50 mM 7 5 Testing a new protein lysate for successful labeling It is important to check that the labeling of the proteins has worked before the sample is used in 2D DIGE The method involves running a small sample of your new labeled lysate on 1 D SDS PAGE gel along with a control lysate that has 20 already been labeled successfully This gel is then scanned for CyDye and the total fluorescence of each labeled sample is compared 1 Label the new protein samples with Cy5 following the instructions in Protein sample labeling with CyDye page 16 Cy5 labeled lysates have negligible cross talk with the Deep Purple dye that might be used later in the experiment 2 Add a volume of each CyDye labeled protein lysate equivalent to 50 ug into a microfuge tube 3 Add an equal volume of 2 x gel loading buffer to the labeled protein lysate 4 Heat the samples at 95 C for 5 minutes to ensure full reduction of the proteins 5 Make serial dilutions of each of the lysates in the 2 x gel loading buffer such as 25 ug 12 5 ug and 6 25 ug Make a 12 5 SDS PAGE gel
7. with gentle agitation for 30 minutes 6 Pour off the wash solution and replace with 500 ml water To make up the working stain solution briefly shake the stain concentrate and add 2 5 ml Deep Purple to make a 1 200 dilution Cover the container to create a dark environment and incubate for 1 hour at room temperature with gentle agitation Note The solution is light sensitive and should be kept out of bright light Note Containers can be wrapped in foil or covered with black plastic It is not necessary to eliminate light completely only to ensure that bright light is significantly reduced Alternatively containers with lids that are a solid colored plastic may be used 7 Pour off the stain and replace with 7 5 v v acetic acid Cover the container to create a dark environment and incubate with gentle agitation for at least 15 minutes 8 Repeat the acetic acid step once The gel can be imaged at this stage NOTE If speed is more important than background levels the gel can be imaged after one acetic acid step Further washes in acetic acid can be performed to reduce the background still further if necessary After imaging the gels can be stored in the dark in 7 5 v v acetic acid at 2 8 C for several weeks This allows for further imaging at a later date if required 23 Table 1 Cells and tissue types tested with 2D DIGE Cell or tissue Lysis buffer Method of cell or tissue Quantity type
8. 0 ul 120 yl 330 ul of rehydration buffer Samples can now be run on an PGphor followed by the 274 dimension on an Ettan Dalt twelve or six For instructions on running 1 dimension IEF and 2 dimension SDS PAGE gels refer to the Ettan DIGE manual or the manuals supplied with the electrophoresis equipment 17 7 Additional information 7 1 Requirements for 2D DIGE protein lysis buffer It is essential that the pH of the protein solution used with a CyDye DIGE Fluor is between pH 8 0 9 0 To ensure that the pH remains between pH 8 0 9 0 a buffer such as Tris Hepes or Bicarbonate should be included in the protein solution at a concentration of approximately 30 m Failure to include a suitable buffer will mean that the pH of the solution will fall below pH 8 0 resulting in little or no protein labeling The Lysis buffer is required to work at 4 C so the pH should be checked when the solution is chilled The protein solution should not contain any added primary amine compounds BEFORE labeling Primary amines such as ampholytes will compete with the proteins for CyDye The result will be fewer CyDye labeled proteins which might affect the data after scanning and spot detection See the section Reagents tested with 2D DIGE page 25 7 2 Requirements for a cell wash buffer A cell wash buffer should not lyse the cells but it should dilute and remove any growth media or reagents that might affect the CyDye labelin
9. 24 7 7 Reagents tested with 2D DIGE Reducing agents DTT 2 mg ml slight reduction in labeling 5 mg ml 2 x reduction in labeling 10 mg ml 10 x reduction in labeling CyDye will bind thiols at increased concentrations TCEP tris 0 5 mM to 1 mM slight reduction in labeling 2 carboxyehtyl 2 mM significant reduction in labeling Phosphine B mercaptoethanol Significantly reduces labeling Detergents Triton X 100 useat1 17 reduction in labeling NP40 upto1 No effect on labeling SDS up to 1 Salts Application of sample during rehydration lt 10 mM recommended Application of sample via cup loading lt 50 mM recommended Buffers Tris Recommend 10 40 mM pH 8 5 pH is very important pH 8 to 9 is optimal Hepes Can cause focusing problems at high concentrations Bicarbonate 5 mM pH 8 5 Acceptable Ches 5 mM pH 9 9 5 Acceptable PPA is a 3 piperidino 5 mM pH 8 Drop in sensitivity as PPA propionamide primary amine 2 D Protein All buffers are compatible with CyDye DIGE Extraction Buffers Fluor minimal dyes 25 Protease inhibitors AEBSF Pefabloc 4 2 aminoethyl Benzolsulphonyl Fluoride Protease Inhibitor Cocktail Complete Aprotinin APMSF 4 amidino phenyl Methane sulphonyl Fluoride EDTA Ethylene Diaminetetra Acetic acid PMSF Phenylmethylsulphony Pepstatin A causes charge trains unless protector reagent is used as above compatib as contains AEBSF e at recommende
10. Amersham CyDye DIGE Fluors minimal dyes for 2D DIGE Reagents for labeling protein with Cy2 Cy3 and Cy5 dyes before 2 Dimensional separation Product Booklet Codes RPKO272 25 8008 60 RPKO273 25 8008 61 RPKO275 25 8008 62 25 8010 65 25 8010 82 25 8010 83 25 8010 85 28 9345 30 Page finder 1 25 Legal Handling 2 1 Safety warnings and precautions 2 2 Storage 2 3 Expiry 3 Components 4 Other materials required 5 6 Protocol Description 6 1 Introduction 6 2 Preparation of a cell lysate compatible with CyDye DIGE minimal labeling 6 3 Reconstitution of CyDye in Dimethylformamide DMF to give a stock solution 6 4 Preparation of CyDye solution used to label proteins 6 5 Minimal labeling a protein sample 6 6 Loading samples onto IPG strips Additional information 7 1 Requirements for 2D DIGE protein lysis buffer 7 2 Requirements for a cell wash buffer 7 3 Cell sonication 7 4 Adjustments of protein sample pH 7 5 Testing a new protein lysate for successful labeling 7 6 Post staining with Deep Purple Total Protein Stain 7 7 Reagents tested with 2D DIGE 8 Troubleshooting guide 9 Related products ON OA ANANA WwW PR N N 12 14 15 16 17 18 18 18 19 20 20 22 25 27 31 1 Legal GE imagination at work and GE monogram are trademarks of General Electric Company Amersham Cy CyDye DeCyder Deep Purple Ettan ImageQuant IPGphor Immobiline Mu
11. E600 Typhoon FLA 9500 DeCyder 2D 7 2 SPN license Ettan Spot Picker Ettan Digester 28 9435 22 28 9435 23 28 9435 24 28 9435 25 28 9435 26 28 9435 27 28 9435 28 11 0033 64 18 1018 06 28 9334 65 80 6485 27 80 6485 08 80 6171 58 28 9374 51 28 9374 52 80 6475 58 80 6179 94 28 9969 43 28 9854 11 18 1145 28 18 1142 68 For Immobile DryStrip gels please refer to the catalogue For more details see Ettan DIGE user manual catalogue and website 31 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan For your local office contact information visit www gelifesciences com contact GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK http www gelifesciences com imagination at work RPKO272PL AG 11 2013 28953163
12. Fluors have an NHS ester reactive group and are designed to covalently attach to the epsilon amino group of lysine of proteins via an amide linkage The quantity of dye added to the sample is limiting in the reaction hence this method is referred to as minimal labeling This ensures that the dyes label approximately 1 2 of the available lysine and then only on a single lysine per protein molecule The three dyes are matched for size and charge such that the three labeled protein samples are all run on the same 2 D gel the same protein labeled with each CyDye will overlay The lysine amino acid in proteins carries a 1 charge at neutral or acidic pH CyDye DIGE Fluors also carry an intrinsic 1 charge which when coupled to the lysine replaces the lysine s 1 charge with its own ensuring that the pl of the protein does not significantly alter CyDye DIGE Fluors when coupled to the protein add approximately 500 Da to the protein s mass in a uniform manner giving an image comparable to equivalent silver stained gels in existing databases 10 Spot picking To spot pick from a gel containing CyDye labeled samples post stain the gel with Deep Purple to ensure that the majority of unlabeled protein is picked to give sufficient protein for MS identification The migration difference between the unlabeled and labeled protein is due to the addition of a single CyDye molecule to the protein This is more significant for low molecular
13. ading buffer 120 mM Tris pH 6 8 20 v v 87 v v Glycerol 4 w v SDS 200 mM DTT and a few grains of Bromophenol Blue 12 5 Acrylamide gel for SE600 32 ml Acrylamide Bis 40 v v 25 ml Tris 1 5 M pH 8 8 1 ml SDS 10 v v 1 ml Ammonium Persulfate 10 w v 40 pl TEMED Make up to 100 ml with distilled water e 1 x SDS electrophoresis running buffer 25 mM Tris 192 mM Glycine 0 2 SDS Store at room temperature 5 Description 2D Fluorescence Difference Gel Electrophoresis 2D DIGE is a method to label proteins with CyDye Fluors that are subsequently separated using 2 Dimensional gel electrophoresis This protocol is specifically designed to work using CyDye DIGE Fluors The 2D DIGE technology is designed to simplify the process of detecting and identifying proteins using the 2 D electrophoresis technique by allowing the separation of up to three different protein samples in the same 2 D gel Each of the three protein samples are labeled with one CyDye After labeling the three samples are mixed together and run on the same Isoelectric focusing IEF and SDS PAGE gel The ability to multiplex different samples on the same gel means that the different samples will be subject to exactly the same 1 and 24 dimension running conditions so the same protein labeled with a CyDye will migrate to the same position on the 2 D gel this helps limit experimental variation Each of the individual samples can then be visuali
14. between low fluorescence glass plates See Ettan DIGE User manual for glass plate recommendations The gel should be made with wells at the top of the gel where the samples will be loaded The SE600 gel system is recommended for this verification 6 Load each protein serial dilution in successive lanes on the gel 7 Run the samples until the Bromophenol Blue dye front has nearly reached the bottom of the gel 8 Scan the gel at the Cy5 wavelength with Typhoon and carry out the statistics in ImageQuant software If the labeling efficiency is comparable to the control CyDye labeled samples can be subjected to 1 and 2 4 dimension electrophoresis 9 If further labeling is required to test a chemical component then the whole process should be repeated starting at point 1 or the 21 sample can be resuspended in the recommended lysis buffer after using the PlusOne 2 D Clean Up Kit from GE Healthcare If labeling of the new lysate appears poor compared to the control sample the reason for this must be determined The pH of the sample should be satisfactory as this has previously been checked with pH test strips The simplest explanation is that less of the new lysate was loaded on the gel than the control lysate This can be tested by post staining the gel for total protein using Deep Purple or a general post stain such as silver staining If an equivalent amount of the new sample and control sample were loaded on the gel this w
15. ching the sides Start with the sonicator set initially at a low setting such as 25 power or 5 um amplitude Increase the sonication gradually so that small white bubbles appear around the tip of the probe this is now at an ideal sonication level When the bubbles appear do not increase the power any further as this will cause the protein sample to froth If the samples do froth briefly microfuge them and then continue sonicating with a reduced power level When the sonication is at the ideal level sonicate for 20 second bursts followed by a 1 minute cooling period Repeat this process five times Alternatively some sonicators have a pulse facility 19 which can be used to achieve the equivalent sonication time Some samples may need further sonication cycles 6 Sonication is complete when the solution appears significantly less cloudy than the starting solution 7 After sonication centrifuge the samples at 12 000 x g for 5 minutes at 4 C Remove the supernatant to a new tube and discard any pellet 8 Samples are now ready for labeling with CyDye 7 4 Adjustment of protein sample pH If the pH of the protein sample is below pH 8 0 do not proceed with labeling with CyDye first increase the pH of the sample In the following example the lysate pH is too low at pH 7 5 ina solution containing 7 M Urea 2 M Thiourea 4 CHAPS and 30 mM Tris 1 Make an identical lysis solution 7 M Urea 2 M Thiourea 4 CHAPS 30
16. ciences com GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 2 Handling 2 1 Safety warnings and precautions Warning For research only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Warning All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In the case of contact with skin or eyes wash immediately with water See material safety data sheet s and or safety statement s for specific advice CAUTION This dye is intensely colored and very reactive Care should be exercised when handling the dye to avoid staining clothing skin and other items The toxicity of Cy 2 Cy3 and Cy5 NHS Esters has not yet been evaluated 2 2 Storage Store at 15 C to 30 C Avoid light store in the dark 2 3 Expiry For expiry date see outer packaging 3 Components RPKO272 25 nmol 5 x 5 nmol of CyDye DIGE Fluor Cy2 minimal dye RPKO273 25 nmol 5 x 5 nmol of CyDye DIGE Fluor Cy3 minimal dye RPKO275 25 nmol
17. concentration Investigate this using the Testing a new protein lysate for successful labeling page 20 Test this using the Testing a new protein lysate for successful labeling section and Post staining with Deep Purple page 22 a Make a new batch of protein lysate reducing the volume of lysis buffer to increase the protein concentration b Increase the ratio of CyDye to protein c Precipitate the proteins and resuspend them in a smaller volume of lysis buffer Always check the pH and concentration of the new sample before labeling Problem The fluorescent signal is weak when scanned on a 2 D gel continued Possible cause Remedies 11 Incorrect fluor to protein 11 400 pmol of fluor per ratio 50 ug of protein is recommended If there is a large concentration of other components which can react with the fluor then more fluor up to 2 nmol per 50 ug of protein can be used 30 9 Related products 2 D Protein Extracti 2 D Protein Extracti 2 D Protein Extracti 2 D Protein Extracti 2 D Protein Extract 2 D Protein Extracti on Buffer Trial Kit on Buffer on Buffer II on Buffer III ion Buffer IV on Buffer V 2 D Protein Extraction Buffer VI IPGphor 3 Isoelectr Multiphor II IPGbox Ettan DALTsix Sepa Ettan DALTsix Sepa SE600 Ruby DIGE gels DIGE Buffer Kit ic Focusing Unit ration Unit 220V ration Unit 115V Low Fluorescence glass plates Ettan Dalt S
18. d concentrations compatib recomme compatib 10 mM compatib recomme compatib recomme 26 e at manufacturer s nded concentrations e between 0 5 mM and e at manufacturer s nded concentrations e at manufacturer s nded concentrations 8 Troubleshooting guide Problem The pH of the protein lysate is less than pH 8 prior to labeling Possible causes Remedies 1 The lysis of the cells has 1 Increase the buffering caused a drop in the pH capacity of the Lysis buffer to 40 mM Tris 50 mM is the recommended maximum for Tris 2 The cell wash buffer was not 2 Increase the pH of the lysis completely removed prior to buffer by the addition of addition of the lysis buffer a small volume of 50 mM NaOH Or add an equal volume of the lysis buffer that is at pH 9 5 Problem The fluorescent signal is weak when scanned on a 2 D gel Possible causes Remedies 1 The fluors after reconstitution 1 Check the expiry date on have a fixed lifetime in CyDye DMF that may have been exceeded 2 The DMF used to reconstitute 2 Always use the 99 8 CyDye was of poor quality or anhydrous DMF to has been opened for longer reconstitute CyDye DIGE than 3 months Fluors Breakdown products of DMF include amines which compete with the protein for the CyDye labeling 3 CyDye has been exposed to 3 Always store CyDye in the light for long periods of time dark 27 Problem The fluorescent signal is weak when scan
19. g process The cell wash buffer should not contain any primary amines A range of cell wash solutions such as 75 mM Phosphate Buffered Saline PBS can be used in conjunction with the DIGE technology as long as their compatibility with the 2D DIGE labeling is evaluated in controlled experiments see Testing a new protein lysate for successful labeling page 20 18 7 3 Cell sonication Sonication with a small micro probe sonicator provides the best and most consistent method for disrupting cells for use in 2D DIGE Sonication will completely disrupt the cells and will also shear the DNA and RNA in the cell resulting in higher quality 2 D gels Presence of large amounts of unsheared nucleic acids can cause vertical streaking in a 2 D gel DNases and RNAses can be added but these may appear as protein spots on the 2 D gel Sonication can be used on many different cell types including bacteria and mammalian cells 1 Clean the probe of the sonicator with 70 v v Ethanol and dry thoroughly with a clean tissue Place a beaker of ice water around the sample tube to keep it cold during sonication If the sample is allowed to heat up in the presence of urea some proteins will be carbamylated which will alter the charge pl of the protein producing charge trains of protein across the gel Ensure that the sonicator microtip is suspended with its tip well below the surface of the liquid in the sample tube but not tou
20. ltiphor Pharmalyte PlusOne and Typhoon are trademarks of GE Healthcare companies 2 D Fluorescence Difference Gel Electrophoresis 2 D Fluorescence Difference Gel Electrophoresis 2D DIGE technology is covered by US patent numbers 6 043 025 6 127 134 and 6 426 190 and equivalent patents and patent applications in other countries and exclusively licensed from Carnegie Mellon University CyDye this product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent numbers 5 569 587 5 627 027 and equivalent patents in other countries The purchase of CyDye DIGE Fluors includes a limited license to use the CyDye DIGE Fluors for internal research and development but not for any commercial purposes A license to use the CyDye DIGE Fluors for commercial purposes is subject to a separate license agreement with GE Healthcare Complete is a trademark of Roche Sigma is a trademark of Sigma Aldrich Co Prefabloc is a trademark of Pentapham Ltd Triton is a trademark of Union Carbide Chemicals and Plastic Company Inc 2002 2013 General Electric Company All rights reserved Previously published 2002 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information http www gelifes
21. ned on a 2 D gel continued Possible causes 4 CyDye has been left out of the 20 C freezer for a long period of time The wrong focal plane has been set on the Typhoon The pH of the protein lysate is less than pH8 Primary amines such as Pharmalyte or ampholytes are present in the labeling reaction competing with the protein for CyDye DTT or other substances such as SDS are present in the labeling reaction at too high a concentration 28 Remedies 4 Always store CyDye at 15 C to 30 C and only remove them for short periods to remove a small aliquot Set the focal plane to 3 mm for gels assembled between standard glass plates or platen for gels placed directly on the platen ncrease the pH of the lysis buffer by the addition of a small volume of 50 mM NaOH Or add an equal volume of the lysis buffer that is at pH 9 5 Omit all exogenous primary amines from the labeling reaction Remove the substances from the labeling reaction if not essential If they are essential test if the reduction in labeling efficiency can be counterbalancedby increasing CyDye Problem The fluorescent signal is weak when scanned on a 2 D gel continued Possible cause 10 There is little or no protein in the protein lysate or less lysate was loaded on the gel The protein lysate concentration is too low i e less than 1 mg ml Remedies 8 10 Continued
22. ould suggest that there is a chemical component in the lysate that is affecting the labeling reaction 7 6 Post staining with Deep Purple Total Protein Stain The fluorescent Deep Purple Total Protein Stain fits into the standard 2D gel electrophoresis workflow and is particularly suitable for use with the 2D DIGE system The recommended workflow involves the matching of Deep Purple post stained preparative gels with CyDye labeled analytical gels Deep Purple has been shown to be compatible with the image analysis softwares and the stain is compatible with manual or automated spot picking and mass spectrometry for protein identification applications 1 Place an appropriate volume of fixation solution into the containers that will be used to process gels The recommended volume of fixation solution required is 20 fold excess of the gel volume 1000 ml for Ettan DALT gels 2 Dismantle the electrophoresis apparatus Remove one glass plate and place the gel attached to the glass plate into fix solution Note Place only one gel in each container The Immobiline DryStrip can be left attached to help with gel orientation 22 3 Incubate in the fixation solution overnight at room temperature with gentle agitation 4 Take the stain out of the 15 C to 30 C freezer and allow to stand at room temperature for 5 10 minutes 5 Pour off the fixation solution and replace with the wash solution 1000 ml for Ettan DALT gels Wash
23. r supernatant to a labeled tube This is the cell lysate to be used for CyDye labeling Discard the pellet Check that the pH of the cell lysate is still at pH 8 5 by spotting 1 ul on a pH indicator strip If the pH of the cell lysate has fallen below pH 8 0 then the pH of the lysate will need to be adjusted before labeling See Adjustment of protein sample pH page 20 Store cell lysates in aliquots at 70 C until protein concentration is to be determined 13 6 3 Reconstitution of CyDye in Dimethylformamide DMF to give a stock solution The dry CyDye must be reconstituted in DMF Each vial of CyDye must be reconstituted in high quality anhydrous DMF specification lt 0 005 H O 99 8 pure open for less than 3 months On reconstitution in DMF the CyDye will give a deep color Cy2 yellow Cy3 red Cy5 blue Displacement of CyDye during manufacture or shipment of the fluors can be recovered to the bottom of the tube by pipetting the DMF down the side of the tube vortexing vigorously and centrifuging The quality of the DMF used in all experiments is critical to ensure that the protein labeling is successful The DMF must be anhydrous and every effort should be used to ensure it is not contaminated with water DMF after opening over a period of time will degrade with amine compounds being produced Amines will react with the NHS ester CyDye reducing the concentration of fluor available for protein labeling If in
24. tock solution and add 3 ul DMF to give 400 pmol CyDye in 1 ul Note Add the DMF first to the microfuge tube followed by CyDye 1 ul of the diluted dye now contains 400 pmol Quantity of CyDye to be used to label a protein lysate It is recommended that 50 ug of protein is labeled with 400 pmol of CyDye In each tube of CyDye there will be a 1 mM CyDye stock solution It is recommended that 400 pmol of CyDye per 50 ug of protein to be labeled between 100 pmol and 1000 pmol per 50 ug of protein can be used If labeling more than 50 ug of protein then the same fluor to protein ratio must be used for all samples on the same gel 15 Examples of CyDye dilutions that are used recommended example is highlighted are shown in Table 1 Table 1 Examples of some widely used CyDye dilutions Volume of stock Volume of added Total volume yl Concentration of CyDye pl DMF ul CyDye pmoles ul Tr 4 amp 5 ao 2 3 5 400 2 2 4 500 1 1 1000 The CyDye working solution is only stable for 1 week at 15 C to 30 C 6 5 Minimal labeling a protein sample The recommended concentration of the protein lysate is between 5 and 10 mg ml Samples containing from 1 mg ml to 20 mg ml have been successfully labeled using the protocol below The amount of CyDye used in the labeling reaction will have to be determined individually for the type of protein sample being analyzed 1 Add a volume of protein sample equivalent to 50 ug to a microfuge tube
25. weight proteins Protein identification CyDye labeling of proteins does not affect the mass spectrometry data used to identify proteins as only 1 2 of lysine residues are labeled on a single protein CyDye labeling of the lysine will only result in the loss of a single trypsin digest site per labeled protein 11 6 Protocol 6 1 Introduction This protocol provides all the information required to use CyDye DIGE Fluors to label proteins for 2 D electrophoresis experiments It is recommended that the protocol is read thoroughly before using the system and that it is followed precisely Reagents tested with 2D DIGE are listed on page 25 26 In the standard labeling protocol proteins are first solubilized in a lysis buffer The protein concentration should then be determined using a standard protein quantitation method CyDye DIGE Fluors are then added to the protein lysate so that 50 ug of protein is labeled with 400 pmol of fluor The reaction is incubated on ice in the dark for thirty minutes When handling proteins it is important to keep them on ice at all times to reduce the effect of proteases and use plastic tubes as many proteins will adhere to glassware The fluorescent properties of Cy2 Cy3 and Cy5 can be adversely affected by exposure to light so it is recommended that all labeling reactions are done in the dark in microfuge tubes and the exposure of protein labeled with CyDye to all light sources is kept to a minimum
26. zed independently by selecting the individual excitation and emission wavelengths for each CyDye when fluorescence scanning Experimental design The experimental design recommended is based on evidence that the experimental variation in a 2 D gel electrophoresis experiment is mostly due to gel to gel variation Running multiple samples on a single gel reduces the number of gels required to produce the same number of data sets The recommended protocol suggests that an internal standard sample be run on all gels within an experiment The standard sample is generated by mixing together an aliquot of all the different samples in an experiment This means that every protein from every sample will be represented in the standard that is present on all the gels The standard sample will increase the confidence in matching between gels and will also allow the generation of accurate spot statistics between gels When using DeCyder 2D software use the experimental design outlined below The example given is for protein samples one derived from a control tissue and one a diseased tissue Mix 1 3 of each of the control and diseased samples together to create a standard sample Label the standard sample with Cy2 Label the remaining 2 3 of the control sample with Cy3 Label the remaining 2 3 of the diseased sample with Cy5 More complex experimental designs can be generated using the standard sample on all gels Minimal labeling CyDye DIGE
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