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<br>MAIN STEPS/TIME TABLE <br>________________________________________ <br>- Solution preparation [2 hours] <br>- Cleaning of aparatus [20 min] <br>- Seperating gel preparation and powing into glasses [30 min] <br>- Waiting for polymerization of seperating gel [1 hour] <br>- Stacking gel preparation and powing onto polymerized gel [30 min] <br>- Waiting for polymerization of stacking gel [1 hour] <br>- Sample preparation [20 min] <br>- Running conditions <br> - for 145 min at 30 mA 120 V through gel <br> - amper is constant <br>- Keeping gel in fixing solution [1.5 hour] <br>- Silver staining and visualization [2 hours] <br>- Coomassie Blue Staning [3 hours] <br>MATERIALS <br>________________________________________ <br>- Vertical Electrophoresis apparatus <br>- Power supply <br>- pH meter <br>- Balance <br>- Silver staining shaker platform <br>- Transilluminator <br>- Graduated cylinder <br>- Spatula (plastic and metal) <br>- Filter paper <br>- Bottle <br>- Pipette (200 uL, 10 mL) <br>- Gloves <br>- Mask <br>- Aluminum foil <br>For Gel preparation <br>- Acrylamide <br>- Bisacrylamide <br>- Deionized Water <br>- Tris base <br>- SDS <br>- APS <br>- TEMED <br>For Silver Stanning <br>- Fixer <br>- %50 ethanol <br>- Pretreatment solution <br>- Silver nitrate <br>- Devoloping solution <br>- Stop solution <br>- Marker: Fermentas / Page Ruler Protein Ladder SM0661 (10-200kDa) <br>SOLUTIONS <br>________________________________________ <br>Running Buffer (10L, 1X) <br>- 25mM Tris, pH 8.3 <br>- 250mM Glycine <br>- 0.1% SDS <br>or <br>- 60 ml 10X stock + 6 ml 10% SDS + 534 ml dH2O <br>----------------------- <br>- 30.3g Tris <br>- 187.7g Glycine <br>- 10g SDS <br>- Final volume 10 L in demijon <br>Staining Solution (Coomassie Blue) ( 1 L, 1 X) <br>- 2 g brilliant blue (R-250) <br>- 450 ml methanol <br>- 450 ml dH2O <br>- 100 ml acetic acid <br>- store @ 24 C <br>Destaining Solution ( 4 L, 1 X) <br>- Methanol:Acetic Acid:dH2O 40:10:50 <br>- Prepare 4.0 L (1.6 L MeOH, 0.4 L AcA, 2.0 L dH2O) in 1 gallon amber bottle, cap tightly <br>- store @ room temp. <br>4X Sample Loading Buffer <br>- 400 mM DTT <br>- 40 mM Tris <br>- 10% Glycerol <br>- 4% SDS <br>- 0.4% Bromophenol blue <br>-------------------------------------- <br>- prepare 15 ml <br>- 925.2 mg DTT <br>- 72.6 mg Tris <br>- adjust pH to 6.8 <br>- 1500 ul Glycerol <br>- 600 mg SDS <br>- 6 mg Bromophenol blue <br>- aliquot 50 x 600 ul <br>- store at -20 C <br>- vortex well <br>30% Acrylimid 1% Bis-acrylimide <br>- prepare in laminar flow <br>- 30 g acrylimide and 1 g bis-acrylimide in 100 ml (toz stock kimyasal icin) <br>- 75 mL acrylimide solution ( 40% stock, Apllichem) and 25 mL bis-acrylimide solution ( 2% stock, Apllichem)to final volume 100 ml (sıvı stock kimyasal icin) <br>- store at 4 C, stable for 1 month <br>Seperating Gel Buffer <br>- 1.5 M Tris pH 8.8 <br>------------------------------------------ <br>- for 100 mL Buffer solution <br>- 18,15 g Tris pH 8.8 <br>- store at room temp. <br>Stacking Gel Buffer <br>- 0.5 M Tris <br>------------------------------------------ <br>- for 50 mL Buffer solution <br>- 3 g Tris pH 6.8 <br>- store at room temp. <br>APS (Ammonium Persulfate) Stocks (100 mg/ml) <br>- dissolve 0.6 g in 6 ml dH2O <br>- aliquot 10 x 600 ul and store @ -20 C <br>1% Bromophenol Blue <br>- 0.01 g in 1 ml 1M Tris, pH 7.0 <br>- store at room temp. in amber bottle <br>CASTING 13% GEL <br>- set heater to 100 C for sample prep step <br> Seperating Gel (30 ml) <br> - 13 ml 30% Acrylimide 1% Bis-acrylimide <br> - 7.5 ml seperating gel buffer <br> - 8,45 ml dH2O <br> - 500 ul %10 SDS <br> - 250 ul APS (initiator of polymerization) <br> - 25 ul TEMED (catalyst of polymerization) <br> - Load 5.4 ml separating gel between glasses <br> Stacking Gel (10 ml) <br> - 1.6 ml 30% Acrylimide 1% Bis-acrylimide <br> - 2.5 ml stacking gel buffer <br> - 5.85 ml dH2O <br> - 100 ul %10 SDS <br> - 15 ul 1% Bromophenol Blue <br> - 50 ul APS (initiator of polymerization) [add after resolving gel is casted] <br> - 10 ul TEMED (catalyst of polymerization) [add after resolving gel is casted] <br> - Load 1.7 ml stacking gel between glasses <br> - Inset the comb <br>- make sure the comb has not been inserted in a tilted way. check from behind the apparatus <br>- load seperating gel <br>- add some butanol or isopropanol before resolving gel solidifies <br>- make sure gel stays on flat surface while solidifies to prevent tilted surface <br>- load stacking gel <br>- if bubbles form in the stacking gel after polymerization, press the plates between hands to push them out <br>SAMPLE PREPARATION AND LOADING <br>- Do not overload the the samples, purity check is difficult with overloaded samples. <br>- Sample volume: 5 ul sample+ 5 ul loading buffer + 10 ul dH2O <br>- vortex loading buffer before use <br>- put samples in heating block (100 C) for 5 min <br>- if possible, do not load into the first and last lanes <br>- load 5 ul marker <br>- load 17 ul samples <br>PREPERATION <br>- check the wire on running apparatus, clean and test <br>RUNNING <br>- never terminate the run early, lighter bands dont separate <br>- 600 ml running buffer is required for each run <br>Running Standards <br>- 5 mA > 3/4 of gel > 25hrs >>> overnight running <br>>> bizim icin 2mA <br>- max: 80 mA for both old and new gel systems <br>- For Lab 103 Tankı <br> - for 145 min at 30 mA 120 V through seperating gel <br> - amper sabit <br>After Run <br>- wash electrophoresis unit after each use <br>- weekly cleaning of power connections recommended to prevent oxidation <br>STAINING <br>- Stain the gel for 30 min <br>DESTAINING <br>- load the tray fully with destaining buffer <br>- do not put too many (over-destaining) or too less (under-staining) paper sheets <br>- destaining takes 2-3 hrs <br>NOTES <br>________________________________________ <br>SEPERATİNG GEL %13 (30 ML) <br>Load 5.5ml from seperating gel between glasses <br>13 ml Acrylamide\bisacrylamide <br>7,3 g acrylamide <br>0,2 g bisarylamide 25 ml d.H2O <br>8.45 ml dH2O <br>7,5 ml 1,5 M Tris-Base pH=8,8 <br>18,15 g Tris-Base <br>100 ml d.H2O pH=8,8 HCI <br>500 ul SDS <br>0,3 g SDS <br>3 ml d.H2O <br>250 ul APS <br>50 mg APS <br>500 ul d.H2O <br>25 ul TEMED <br>Stacking Gel (10 ml) <br>Load 1.7 from stacking gel between glasses <br>1.6 ml Acrylamide/bisacrylamide <br>7,3 g acrylamide (T=%30, C=%2,67) <br>0,2 g bisarylamide 25 ml d.H2O <br>5.85 ml dH2O <br>2,5 ml 0,5 MTris-Base pH=6,8 <br>3 g Tris-Base <br>50 ml d.H2O pH=6,8 HCI <br>100 ul SDS <br>0,3 g SDS <br>3 ml d.H2O <br>50 ul APS <br>50 mg APS <br>500 ul d.H2O <br>10 ul TEMED <br>TROUBLESHOOTINGS <br>Smear / GuHCl / Urea <br>Normally having 6M GuHCl or Urea will cause a lot of band smearing and will not produce sharp bands on SDS -PAGE. <br>Alcohol or Acetone ppt. is suggested for proteins denatured by GuHCl or Urea for running on gels. <br>SDS <br>SDS should be added to all gels and the tank buffer at 0.1%. You can replace half the SDS in the Cathode buffer with 25 mg/l CBB-G250, then you can watch the protein bands during electrophoresis (Schägger et al., Anal. Biochem. 173 (1988) 201-5) <br>Separation of proteins with small MW difference <br>- Increase the gel % to upto 20% <br>- A longer SDS-PAGE apparatus, that will be of assistance as you can actually run the proteins out to a greater extent. <br>- A gradient gel is usefull if you want to separate small and large proteins on the same gel. <br>- Prepare the samples as usual, i.e. add Laemmli buffer or whatever you use, but leave out the SDS from the gel while preparing it. Include SDS in the running buffer however. I have seen some appr. 10-kDa proteins that differ by 2 amino acids separate quite well under these conditions. The gel % around 15-20... <br>Ghost (Double) Bands <br>Degradation <br>PMSF related > protease action > degradation <br>PMSF is not a very efficient protease inhibitor and if I am not mistaken it only works against serine proteases. <br>As a remedy, we can purchase and use a better protease inhibitor... <br>Oxidation Related <br>Reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol cleave disulfide bonds into free sulfhydryl (SH) groups to allow proteins to unfold completely. However, the reducing agent can be oxidized during sample heating which may allow these disulfide bonds to reform, leading to the appearance of ghost bands in the high molecular weight area or precipitation at the sample application point. <br>Blocking the reduced SH groups can prevent disulfide bonds from reforming. One common way to do this is to alkylate with iodoacetamide. Iodoacetamide also alkylates residual DTT to prevent point-streaking and other artifacts in horizontal flatbed gels. The recommended amount of iodoacetamide is 2.0-2.5% (w/v) in the sample. The iodoacetamide should be added after boiling the reduced sample, but prior to loading the sample onto the SDS-PAGE gel. Alternatively, for first-dimension IPG strips, perform a second equilibration step for the IPG strips with an iodoacetamide solution in SDS equilibration buffer (without DTT). The alkylation reaction should take 15-20 minutes at room temperature.

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