Core Facility (Houghton'04)


2 D Electrophoresis
1st Dimension
Contact: Debby Walthall or Hyuk-Kyu Seoh Rm: NSC 338/NSC 516 Tel: (404) 413-5363 or (404) 413-5379

2D electrophoresis manuals:
Sample Prep
2D Cleanup
2D Quant

1st Dimension topics:
Supply List
Reasons for doing rehydration and IEF together or separately
Strip rehydration and IEF separately

Strip rehydration and IEF at same time
IEF programs
Cleaning 1st Dimension
Buffers and Solutions for 1st Dimension

General Warnings:
Every time you use the 2-D equipment, you MUST sign the logbook.

GE Amersham Manuals:
Amersham 2-D electrophoresis Manual
Ettan DIGE System User Manual

Equipment for 1st-dimension:
IPGphor Isoelectric Focusing System
IPGphor Rehydration Tray
IPGphor Cup Loading strip holder (can choose 7, 11, 13, 18 or 24 cm)
IPG strip (Immobiline DryStrip gels) - can choose 7, 11, 13, 18 or 24 cm; many choices of pH ranges – see below.

IEF is an electrophoretic method that separates proteins according to their isoelectric points (pI). The net charge of a protein is the sum of all the negative and positive charges of its amino acid side chains and amino- and carboxyl- termini. The isoelectric point is the specific pH at which the net charge of the protein is zero. In a pH gradient, under the influence of an electric field, a protein will move to the position in the gradient where its net charge is zero. IEF performed under denaturing conditions gives the highest resolution and the cleanest results. First dimension separation requires selecting a 1st dimension pH range appropriate for the samples as well as a suitable sample application method. The procedure includes IPG strip rehydration, sample application and isoelectric focusing. The protocols selected are specific for your sample and the equipment you have.

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 – 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. 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. We HIGHLY recommend using these kits especially the 2D Cleanup kit.

Notes:
• Buy strips from Amersham. Strips and boats come in different sizes (cm lengths) and different pH ranges – what you use depends on what you want; there are broad pH range strips and narrow, specific range pH strips (ex. 4.5 –5.5). Usually you want to start out with a broad pH range and then as you figure out what you want, you can go to a narrow range and get better resolution.
• If running CyDyes, protect strips from light at all stages.
• Don’t touch strip with hands once plastic backing off.
• Note + end (old strips have a point on + end; new strips actually have a “+” and a barcode on the + end).
• You want 1/8 – 1/4 of volume to be sample + 2X sample buffer. Remaining volume is for the rehydration buffer and depends on strip length. You want a high concentration of DTT or Destreak.
• Rehydrate – swells to ~1 mm and takes 12 – 16 hrs. Can be done along with IEF or separately.
• If doing rehydration and IEF at the same time, adjust time so that run doesn’t end in the middle of the night. When run is over, gel can sit no more than an hour; after that the proteins start to diffuse and your spots aren’t clean. Add an additional step at the end (Rollin’s program) that is optional so run doesn’t end in the middle of the night.
• You can freeze the strips until you can run the 2nd dimension, but the gel has to be placed (no buffer) into a plastic tube (equilibration tube or tube that cups come in) and then goes into –80oC.Boats (2 parts – holder and lid).
• Boat size must match the strip size.
• Clean 1st dimension equipment with a nonionic detergent (Strip holder cleaning solution.). Clean after use and then again before use.

Notes from Roberto:
• Proteins will precipitate if they sit too long after IEF. Be sure to do a quick freeze in liquid N2 and store at –80oC if not running 2nd Dimension right away.
• You could run 24 cm strips and then cut into 3 pieces to run 2nd Dimension on small vertical gels. This would be a cheap and quick way to view your proteins and troubleshoot before running large gels.
• Use bar code to keep track of your samples.
• Use SDS (or can dilute any SDS with rehydration solution; concentration can’t be higher than 0.005%). Don’t use Triton X – interferes with Mass spec analysis.
• Need to optimize IPG buffer concentration. 0.5% is optimal, but you can go up to 2%. If your spots have a horizontal tail – try increasing the IPG buffer %.
• Need to optimize DTT concentration also. DTT tends to cause streaking in high pH. Destreak has better buffering in the high pH ranges. Destreak is used in the rehydration buffer and has a reduced concentration of DTT (8 mg instead of 10 mg).
• Thiourea is used for very hydrophobic proteins and high MW samples.
• Carbamylation – DO NOT HEAT urea ever (not over 30oC). Also over time, urea will start to degrade, so store –20oC for 3 months (book recommends 6 months).
• Dry strip fluid is mineral oil. It’s clean and no ionic charge.
• 1st Dimension manifold – highly abundant proteins
• New manifold. Cup loading – can’t load high concentrations of proteins because only allows a low volume to be loaded. Paper filter loading – large surface so can load higher volumes (350 ml) and therefore a higher concentration of proteins.

Read the Amersham manual for details about (we have an IPGphor IEF system):
•Isoelectric focusing background.
•pH gradient selection
•Sample application method selection
•Rehydration solution – components and why each one is in solution
•Types of IPG strips available and pH ranges
•IEF guidelines
•IPGphor specific information:
•IPG rehydration + sample setup
•IPG rehydration and sample run separately
•Sample prep using cup loading
•Guidelines for setting up the IPGphor protocol
•Running a protocol (including a table for protocols for specific pH and length strips
•Troubleshooting

Key:
LB     Lysis Buffer
SB   2X Sample Buffer
RB    Rehydration Buffer
EB    Equilibration Buffer

IPG strip can either be rehydrated at the same time as IEF or rehydrated o/n and then IEF done the next day (or o/n).
Reasons for doing at the same time:
•You can load more protein and also more volume which is helpful for dilute samples.
•Simpler
Reasons for doing separately:
•If you are worried about proteolysis or protein modifications occurring o/n.
•Better protein focusing, especially in the high pH range

Gel Rehydration and IEF at same time. Anupama
Protein sample + 2X Sample buffer (SB) + rehydration buffer (RB). See Start/Decisions to be made for amount of protein sample; add equal volume 2X SB, incubate on ice for 30 min.; bring volume up to 340 with RB for 18 cm strips or 250 ul for 13 cm strips. See manual for volumes used for other size strips. 2X sample/1X rehydration solution can be stored in aliquots at –20oC for 6 months. Sample buffer and Rehydration buffer are made fresh from the 2X Sample/1X rehydration solution before each 1st D run.

Gel Rehydration and IEF done separately. Rollin/Shelby (Pierce Lab)
Gel strip + RB o/n (20 – 30 hrs). Protein sample + 2X SB, incubate on ice for 30 min.; bring up to 100 ul w/ RB, apply to gel on either end of strip so solution goes under strip not on top of plastic backing. 2X sample/1Xrehydration solution can be stored in aliquots at –20oC for 6 months. Sample buffer and Rehydration buffer are made fresh from the 2X Sample/1X rehydration solution before each 1st D run.

IEF
Run Gel Rehydration and IEF done separately Rollin/Shelby
IPG strip rehydration with reswelling tray and Sample cup
Rollin and Shelby use 24 cm strips, so the amounts listed are for the long strips. Using toothbrush, clean reswelling tray with IPG detergent (or other non-ionic detergent). Rinse thoroughly with ddH20 and air dry (if needed, use crewipes to dry). Clean after use and also just before use.; but the boats/manifold must be dry before running.
1. Rollin and Shelby use 24 cm strips, so the amounts listed are for the long strips.
2. Using toothbrush, clean reswelling tray with IPG detergent (or other non-ionic detergent). Rinse thoroughly with ddH20 and air dry (if needed, use crewipes to dry). Clean after use and also just before use.; but the boats/manifold must be dry before running.
3. Remember to always wear nitrile gloves. Leave strips in freezer until ready to load.
4. Remove appropriate amount of Destreak rehydration solution from -20C. Bottle contains 3ml of solution. 450ul (for 24 cm strip) required per well. Once thawed, vortex vigorously until white solid (urea) dissolves. Allow to come to RT.
5. Add IPG buffer (or other ampholytes) to Destreak solution to 0.5% v/v (15ul to 3ml, 60ul to 6ml). Note: Ampholyte range should match that of the strip.
6. For a right –handed person, orient the reswelling tray so that the acidic (+) end is to the left. Acidic end has the little circular well. Pipet 450ul of rehydration solution into the appropriate number of wells. Note: Some find that applying rehydration solution toward cathodic (-) end of well results in more complete rehydration.
7. Load strips. Remove strips one at a time and load. The following description uses your gloved hands to manipulate the strip. You can also use 2 forceps. Hold acidic end (the one with the bar code) between thumb and index finger of left hand so that the protective cover is facing away from left hand. With right hand, gently peel off protective cover. Not as easy as it sounds, try flicking the end of the strip with your thumb. This will usually loosen the strip enough to pull apart. Again with right hand, grasp basic end backing between thumb and index finger so that when the strip is flipped, the gel side is facing down and the plastic backing is up. Lower the strip so that acidic end contacts pool of rehydration buffer. Gently slide the strip back and forth across the solution, making sure that gel is wet before contacting any part of tray that is dry. Lower and release basic end so that acid end completely crosses well at acidic end
8. The above description is one way to hold and manipulate the strip. You will need to develop one that you are comfortable with. Just keep track of which side of the strip has the gel. The gel side needs to be down. You need to be sure that the gel never touches the dry tray well.
9. Cover each strip with 3.4ml of Drystrip cover fluid (mineral oil).
10. Insert cover, gently move tray to place of incubation, and balance with black nobs.
11. Incubate strips for 20-30 hours at room temp.

Sample Prep and Loading IPGphor
1. Rollin and Shelby use 24 cm strips, so the amounts listed are for the long strips.
2. Step 10 amounts are for CyDye samples. “Reduced sample” term is also in the step. This is described in the manual as “preparing the sample”; Rollin and Shelby call this step Reduction (holdover from 1D gels and reducing the protein before loading). No matter what you call it – this is the step where the sample is incubated with the 2X sample buffer for 30 min. on ice.
3. Sample Prep/Reduction. 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. Incubate on ice for 30 min.
4. Using treezers, grasp the strip’s basic end backing (away from bar code). Lift, flip, and place in ceramic boat so that acidic (the end with the + and barcode) is pointed toward pointed end of the boat and the cathodic end of the gel is at the square end of the boat. Make sure the gel surface is up.
5. Cover each rehydrated strip with 4.4ml Drystrip cover fluid (mineral oil).
6. Place electrode pads moistened with 150ul ddH20 on cathodic and acidic end on gel, not the gel backing.
7. Place electrodes on electrode pads, several mm from the ends of the gel. Ensure that the electrode is over the gel, not the gel backing.
8. Place sample cup several mm from the acidic end electrode pad. Ensure that the cut feet contact the bottom of the boat and that they do not contact the boat’s guides. Observe at eye level to make sure.
9. Fill each sample cup with 100ul of Drystrip cover fluid. Allow gels to sit for 10 min.
10. Add 28ul RB to each reduced sample (total volume now 100ul; 10 ul control + 10 ul sample + 10 ul standard + 3 ul lysine + 3 ul CyDyes + 36 ul SB + 28 ul RB). Bring preparative sample up to 100ul with RB. Vortex each and spin down.
11. Ensure that cups are not leaking by making sure that level of Drystrip cover fluid in each cup has not dropped.
12. Add entire sample contents (100ul) to sample cup.
13. Turn on IPGphor, back right. See below for instructions about navigating the menu.
14. This machine holds 12 strips, but if fewer, just spread evenly across the surface. Place boats on IEF platform. Ensure that boat electrodes contact platform electrodes (the gold part). Place strips lengthways with positive end towards 18 cm or 13 cm (size of strip and boat) mark on left side of surface. Line strips so that they are even with the 18 cm or 13 cm mark. This alignment will have the – end fall on the appropriate part of the gold surface. Place covers over boats (tab down) and close hood.
15. Cover with foil if using CyDyes.
16. Return remaining pooled sample to -70.
17. After run completed, remove strips from IPGPhor and wipe gold surface off with a Kimwipe. Remove lid from boat.
18. Using forceps, lift strip out of boat. Touch 1 end of strip to boat and let oil drain. Touch 1 end to a piece of filter paper (Whatman 1) to blot oil. Place boat in ddH2O until ready to clean. Do not let the buffers dry onto the boat.
19. Slide strips into plastic equilibration tubes with gel side up and plastic backing touching the tube side. Cap tube and label tube. Store strips at –70oC.
20. OR You can proceed directly to strip equilibration and 2nd dimension.

Sample Prep, Gel Rehydration and IEF at same time. Anupama (Houghton lab)
1. Anupama uses 18 – 13 cm strips, so her amounts are for these strips.
2. 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. Incubate on ice for 30 min.
3. Add 340 ul/250 ul (prepared sample + 2X sample buffer) + rehydration buffer to center of boat (don’t worry about bubbles).
4. Hold strip with gloved hand by ends. Flick end away from + to separate 2 pieces (the gel and the plastic cover). Hold the hard plastic (gel) with the left hand and facing up. Hold the thin plastic facing down with your right hand and pull it away from the gel/hard plastic. Don’t let thin plastic go back onto gel. Toss thin plastic.
5. Hold gel with your right hand at the end away from the barcode - turn the boat so that the strip + end will end up on the + end of the boat. (If you are using OLD strips, they do not have a + end marked; the pointed end is the +).
6. Place one end onto the rehydration soln already in the middle of the boat (kind of floppy, so be careful). Make sure you haven’t flipped the strip over. You want the gel next to the soln. and the hard plastic on top. Drag strip back and forth to spread the soln. over the length of the boat, moving towards the left. When the strip and soln. reach the end to the left, use your left hand to carefully lift the left end of the boat so that the soln. starts moving some to the right. At the same time, lower the strip onto the soln. – you just can’t move back and forth in quite the same way. Before dropping the right end, push the left end all the way to the end of the boat, then drop the right end. (You go through all this back and forth and lifting so that the dry gel doesn’t ever touch the boat without soln.).
7. Check the strip for air bubbles. If there are any, using the forceps, push on the air bubble – it will just squish out the side of the strip. Don’t worry about 1 end being more blue (and maybe more protein) than the other end. Since the whole theory behind 1st dimension is reaching the spot for 0 charge, the proteins will still go to the correct pI even if not spread evenly.
8. Cover gel evenly with 1 ml of Dry Strip Cover fluid. Just drip the fluid across the strip. Put on boat lid – tabs go inside/down.
9. Turn on IPGphor, back right. See below for instructions about navigating the menu. This machine holds 12 strips, but if fewer, just spread evenly across the surface. Place strips lengthways with positive end towards 18 cm (size of strip and boat) mark on left side of surface. Line strips so that they are even with the 18 cm mark.
10. Close lid. Be careful that lid is really closed. Sometimes you think it is closed and when you go back to check the run, it has stopped and has an error message. You might want to go check the run after 30 min. or so, to make sure everything is running fine.
11. Cover with foil if using CyDyes.
12. After run completed, remove strips from IPGPhor and wipe gold surface off with a Kimwipe. Remove lid from boat.
13. Using forceps, lift strip out of boat. Touch 1 end of strip to boat and let oil drain. Touch 1 end to a piece of filter paper (Whatman 1) to blot oil. Place boat in ddH2O until ready to clean. Do not let the buffers dry onto the boat.
14. Slide strips into plastic equilibration tubes with gel side up and plastic backing touching the tube side. Cap tube and label tube. Store strips at –70oC.
15. OR you can proceed directly to strip equilibration and 2nd dimension.

IPGphor Programs
1. I highly recommend adding an extra step at the end of any program. You do not want the strip to just sit without some current for any length of time before either freezing or going to Equilibration steps. This extra step also gives you some control over estimating when the 1st D will be done. This step is NOT essential to focusing. As soon as the program goes to the extra step (S5 or S6/S7 depending on whether you do separate rehydration; from here on I will just say S5/S6/S7 and you decide which is appropriate for your run), you can stop the program. You do not have to go the full 3 hours.
2. A program is listed below, but this is to show you how to manipulate the buttons and make changes in the program. . Each strip length and pH range has different steps and different times/voltages etc. See GE manual pg. 66 – 69. There are several choices for some of the steps. These choices depend on whether you are using the cup loading method or the combined rehydration/IEF method.
3. Anupama adds an additional step after rehydration. Step and Hold; 200 V; 1 hr.
4. When reading the table, don’t forget that these steps start with the IEF steps NOT the rehydration step.
5. Suggestion. Go ahead and setup multiple programs for the different strips labeled with the strip name so that you are ready to go.
6. Turn machine on, stitch is back right hand side. Protocol #1 should be displayed after machine goes through diagnostics.
7. IEF is done at 20oC and 50 uA/strip.
8. Edit. Arrow up to the Protocol you need.
9. Use right arrow to move cursor (under the #1 of S1) to hrs. Use up and down arrows to increase or decrease time. Use left arrow to move cursor back to the #1 in S1 to proceed to the next step. If you don’t go back to S1, the arrows will change other values.
10. Once the cursor is under S1, move onto the next step using the up Arrow.
11. Adding Rehydration step.
S1              rehydration         30 V          12 – 16 hrs.
12. Adding Hold step (S5/S6/S7)
S5/S6/S7                  Step-n-hold         500V          3 hr
13. You can stop the run anytime once it gets to S5/S6/S7
14. Keep doing Arrow up for Steps 7 – 9
S7 – S9                all settings (time, voltage) set at 0
15. Start. Select # of strips. Start.
16. Run time depends on # of strips and purity of sample. # of strips goes up – run time increases. Dirty samples will also take longer. The instrument will give you an ~ run time, this is usually a couple hours long.

Cleaning
1. Boats. As you remove the strips after the run, place the boats in a tray of dH2O. Do not let the buffers dry out in boat. Wash boats with a soft toothbrush, using IPGphore strip holder cleaning soln. Wash well. Do not use any other soap. Rinse with dH2O until not soapy anymore.
2. 1st dimension apparatus. Wipe surface with Kimwipe after run.

Buffers and Solutions
Read the Amersham manual for basic solutions
• Appendix 1 has solutions and buffers used to run the gels.

Following is the Lysis buffer that Rollin uses to resuspend his protein samples:
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.

Lysis buffer
30mM Tris, 7M urea, 2M thiourea, 4% CHAPS

13.2 ml deionized urea/thiourea solution
450 ml 1M Tris, not pH’d
150 ml nuclease mix
300 ml protease inhibitor
600 mg CHAPS
pH to 8.6 with HCL

Bring volume up to 15ml
Aliquot 950 ml/tube and store at -70oC

The 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. If using CyDyes, use pharmalyte, otherwise use IPG buffer. 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 solution #1:
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 #2:
7M Urea, 2 M thiourea, 4% CHAPS

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.
** I had trouble getting the urea dissolved when I used dry urea.

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 donít use ñ 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.

This information is given as a guide to the facilities and instrumentation available in the DNA/Protein Core facility at Georgia State University. If you have any concerns or thoughts about the content of this website please contact: John Houghton (404) 413-5390