Tim Heuer
4/12/98
Strategic Planning: Protein gel electrophoresis is used to analyzeprotein samples, and under denaturing conditions can be used to purifyspecific components of a mixture that contains more than one protein. Likenucleic acid electrophoresis, the charge to mass ratio of each proteindetermines its migration rate through the gel. Because the carbon backboneof protein molecules is not negatively charged, negative charge is providedby the inclusion of sodium dodecyl sulfate (SDS) in the loading, gel, andelectrophoresis buffers. The negatively charged SDS binds to the proteinbackbone and causes unfolding of the protein. The amount of SDS bound toeach protein is proportional to its molecular weight, and the rate of migrationthrough the gel is proportional to the molecular weight by a log-linearrelationship. However, because very small proteins, less than about 10KD, do not bind SDS well, small proteins are more difficult to resolve,and therefore require modified electrophoresis conditions. Electrophoresisof covalently joined protein-nucleic acid fusions can also require modifiedconditions for optimal resolution of different species. This arises fromthe fact that the nucleic acid component often contributes the vast majorityof both the molecular weight and negative charge of fusion molecules, therebyaltering the electrophoretic properties of these molecules. I will firstdiscuss preparing and running what I refer to as a standard protein gel.This standard gel is the system routinely used for electrophoresis of mostprotein samples. I will then discuss modifications that are used for betterresolution of small proteins or peptides, and also modifications for electrophoresisof nucleic acid-protein fusions.
Protocols:
1. Standard Protein-Gel Electrophoresis
Materials
PREPARING A PROTEIN GEL: Threebasic reagents are required that can be prepared from the list of materialsabove: (1) acrylamide gel buffers, (2) electrophoresis buffer, and (3)sample loading buffer.
The first step is to prepare the acrylamidegel. I almost always prepare and run protein gels using 20 cm length glassplates, and will occasionally run them using theHoffer mini-gel system.Standard protein gels are typically composed of two layers (Figure 1).The top-most layer is referred to as the stacking gel, and it comprisesabout 10-20% of the gel height. The stacking layer contains a low percentageof acylamide, typically 3.5-4.0%, and is buffered at pH 6.8. The lowerlayer of acrylamide, which comprises the remaining portion of the gel,is the separating or resolving gel. The acrylamide concentration of theseparating gel varies according to the samples to be run. Commonly, valuesof 8-15% acrylamide are used. The pH of the separating gel is 8.8. Thedifference in pH and acrylamide concentration at the stacking and separatinggel interface functions to compress the sample at the interface and providesbetter resolution and sharper bands in the separating gel. Some peoplechoose to omit the stacking gel, however I personally do not suggest this.
The proteingel is prepared in a manner very similar to nucleic acid polyacrylamidegels, however when pouring a gel that contains a stacking gel, a bottomspacer must be used, and the gel is poured in the vertical position. Theacrylamide solutions are prepared using a stock of 30% acrylamide (37.5:1acrylamide: bisacrylamide) diluted to the appropriate concentration in1X stacking/separating gel buffer. To provide a smooth surface and interfaceat the top of the separating gel, isopropanol is placed above the gel whileit polymerizes. After the gel is completely polymerized, the isopropanolis poured off, and the top of the gel is rinsed with deionized water. Ithen use Whatman paper to carefully soak up any excess water from boththe gel surface and glass plates. With the comb in position at the topof the glass plates I pour the stacking gel on top of the separating gel.This is most easily done by pipeting the acrylamide solution into the gel.
SETTING UP A PROTEIN GEL:Theprimary note to make here is that in addition to rinsing the wells, asis done for nucleic acid gels, the space at the bottom of the gel thatis created by removing the bottom spacer must be rinsed with a syringeneedle to flush any air bubbles out that would interfere with electrophoresis.For this purpose, I have a syringe needle that I have bent so that it canreach up into the crevice. A final important note is that the gel runningbuffer is a tris-glycine buffer that is different from the buffer usedto prepare the gel. It is used at a 1X concentration.
RUNNING SAMPLES ON A PROTEIN GEL:As one might expect, running samples on a protein gel is very similar torunning samples on a nucleic acid gel. The sample is prepared by dilutingit into a concentrated sample loading buffer so that the loading bufferis at a final concentration of 1X. The samples are then heated at 900Cfor 3 minutes and then loaded onto the gel, which is not pre-run priorto sample loading. Protein gels are run more slowly than nucleic acid gels,and consequently may require more time. A gel 20 cm in length generallyruns for about 3-4 hours. The time required is of course variable dependingon the nature of the sample. The samples are run into the stacking gelat about 2-3 watts for 30 minutes or so, after which the power is increasedto 8-10 watts until completion. I always include on the gel a lane thatcontains pre-stained molecular weight markers, which serve two purposes:(1) they allow you to monitor the gel, providing an indicator of how thegel is running, and (2) provide molecular weight indicators so that youcan determine how long to run the gel.
FIXING AND DRYING PROTEIN GELS:Upon completion of electrophoresis the gel is taken apart like a nucleicacid gel. The stacking gel is removed, however, be sure to check it forradioactivity, and dispose of it accordingly. If the gel contains 32Plabeled samples it can be dried directly as you would a nucleic acid gel.If the gel contains 35S-methionine samples the gel should befixed and then washed in a patented solution called "amplify", availablefrom Amersham, which will significantly shorten your exposure time to autoradiographyfilm. To fix the gel it is washed for 20 minutes in a solution of: 50%methanol, 10% acetic acid, and 40% water. The gel is rinsed in water, andthen washed in the "amplify" solution for 20 minutes, after which it isagain rinsed with water. The Amplify solution can be saved and reused 3-4times, or more if there is not a lot of free methione that diffuses intothe solution. With repeated use, this free methionine will cause an increasein the backgound level of your gel. The gel is transferred to saran wrapand then to Whatman paper, and finally it is dried under vacuum. Gels thatcontain 32P samples are exposed while covered with saran wrap.However, the saran wrap must be removed from gels that contain 35Ssamples. It is very important to apply a thin covering of baby powder tothe surface of these gels so that the autoradiography film does not adhereto the gel. I apply it, and wipe of any excess, with a kimwipe. Non-radioactivegels are fixed and stained with either coomassie blue, or silver stains.These protocols are different, but since we do not currently run thesetypes of gels I will not cover them. If anyone ever finds themselves needingto stain a non-radioactive gel I would be happy to provide a protocol atthat time.
RECIPES:
| 1. Stacking Gel Buffer 125 mM Tris-HCl; pH 6.8 0.1% SDS | To make a 4X Stock (500 ml): 30.35 g Tris base; pH'd to 6.8 with HCl. 20.0 ml 10% SDS (or 2.0 g solid) ~400 ml of ddH2O |
| 2. Separating Gel Buffer 375 mM Tris-HCl; pH 8.8 0.1% SDS 20.0 ml | To make a 4X Stock (500ml) 91.0 g Tris base; pH'd to 8.8 with HCl 10% SDS (or 2.0 g solid) ~350 ml ddH2O |
| 3.Electrophoresis Buffer 25 mM Tris; pH 8.3 250 mM Glycine 0.1% SDS pH = 8.3 without adjustment | To make a 5X stock (1L): 15.1 g Tris base 72.0 g Glycine 5.0 g SDS ~850 ml ddH2O |
| 4. Protein Gel Sample Loading Buffer 50 mM Tris-HCl; pH 6.8 2% SDS 10% Glycerol 1% b-Mercaptoethanol 12.5 mM EDTA 0.02 % Bromophenol Blue | To make a 4X Stock (10 ml) 2.0 ml 1M Tris-HCl; pH 6.8 0.8 g SDS 4.0 ml 10% Glycerol 0.4 ml 14.7 M BME 1.0 ml 0.5 M EDTA 8 mg Bromophenol Blue |
STRATEGY: I use this gel systemfor resolving peptides, and importantly, for resolving translation reactionsamples. Although the RNA-protein fusion molecules are much greater than10 KD, I have found this system to give the sharpest bands, and best resolutionof both the unfused peptide and the RNA-peptide fusion. With the currentpool, 232 nucleotide RNA transcript that encodes a 7.4 KD peptide, I usean 8.5% separating gel, and a 4% stacking gel. This gel system is basedupon that of Shägger and von Jagow (Analytical Biochemistry 1987.166, 368-379), however, If you look up the reference paper you willrealize I have modified it slightly. Namely, I have simplified it by usinga single electrophoresis buffer for both the anode and cathode reservoirs,and I use a acrylamide:bisacrylamide ratio of 37.5:1.
Materials
PREPARING THE GEL: Technically,this gel is prepared as described above. Where it differs is in the buffersused for preparation of the gel and for electrophoresis. The recipes aredescribed in detail below. I use a very shallow stacking gel that comprisesabout 5% of the total gel height. Sample loading buffer, and sample preparationis the same as described above.
RECIPES:
| 1. Gel Buffer: 1.0 M Tris-HCl, pH 8.45 0.1% SDS | To make a 3X Stock (500ml) 181.6 g Tris base ~38 ml 12.1 M HCl 1.5 g SDS ~340 ml ddH2O |
| 2. Electrophoresis Buffer: 100 mM Tris pH = 8.25 100 mM Tricine 0.1% SDS | To make a 10X Stock (500 ml) 60.56 g Tris base 89.60 g Tricine 5.0 g SDS ~400 ml ddH2O pH = 8.25 without adjustment |
4. 4% Stacking Gel (7.5 ml):
1.0 ml 30% Acrylamide
2.5 ml 3X Gel Buffer
2.0 ml 50% Glycerol
2.0 ml ddH2O
RUNNING THE GEL: The gel isrun as described above. I run 2 ul of a 12.5 ul translation reaction onthe gel ( plus 4 ul of gel loading buffer). I have begun to include 0.1%Triton X-100 and 2M Urea in my sample loading buffer because I seem tohave fewer things stuck at the top of the separating gel. I generally runthe sample into the stacking gel at 2 watts for 20 min., followed for 20minutes at 6 watts. I then increase the power to 8 watts and run the gelfor about 3 hours. The bromophenol blue does not need to run to the bottom.I like to run it about 60-75% to the bottom.
FIXING AND DRYING THE GEL: Thesame as described above. An overnight exposure of the gel to autoradiographyis sufficient. Exposure to the phosphorimager requires about a 2-day exposure.
3. TBE-Urea gels: Electrophoresisof Nucleic Acid-Protein Fusions.
STRATEGY: TBE-urea gels (nucleicacid gels) can be used for samples that contain nucleic-acid-protein fusionsto give very good resolution of protein and non-protein conjugates. Inthis system the negative charge of those molecules that enter the gel isprovided by the attached nucleic acid. For this type of gel the sampleloading buffer used is the standard urea loading buffer used for nucleicacids. This system works best if the sample is not a complex mixture ofmany proteins that can form high molecular weight aggregates. Therefore,I have not found it to work very well with crude translation reaction samples.Recently though, I have had moderate success by adding 0.1% triton X-100to both the gel buffer and sample loading buffer. Nonetheless, I currentlydo not use this type of gel for analyzing fusion samples, but rather theSDS-tricine system discussed above. I generally use "protein-gel acrylamide"stocks to prepare these gels because the higher acrylamide:bisacrylamideratio (37.5:1) usually works better with these samples. I prepare the acrylamidesolution to contain 8 M urea, 1X TBE, and the appropriate concentrationof acrylamide. I have generally run gels ranging from 6-10% acrylamide.The current pool generates fusions that are about 94 KD, ~87 KD nucleicacid, and 7.4 KD peptide. For these molecules I would use around a 6 %gel. I have run many gels of this type, and I would be happy to discussdetails and customization for a specific application.