4/12/98Proper Planning: Protein gel electrophoresis can be used to analyzeprotein samples, and under denaturing conditions may be used to purifyspecific aspects of a combination which contains several protein. Likenucleic acidity electrophoresis, the charge to mass ratio of every proteindetermines its migration rate with the gel. Since the carbon backboneof protein molecules isn't negatively billed, negative charge is providedby the inclusion of sodium dodecyl sulfate (SDS) within the loading, gel, andelectrophoresis buffers. The negatively billed SDS binds towards the proteinbackbone and results in unfolding from the protein. The quantity of SDS bound toeach proteins are proportional to the molecular weight, and also the rate of migrationthrough the gel is proportional towards the molecular weight with a log-linearrelationship. However, because really small proteins, under about 10KD, don't bind SDS well, small proteins tend to be more hard to resolve,and for that reason require modified electrophoresis conditions. Electrophoresisof covalently became a member of protein-nucleic acidity fusions may also require modifiedconditions for optimal resolution of various species. This arises fromthe proven fact that the nucleic acidity component frequently contributes the vast majorityof both molecular weight and negative control of fusion molecules, therebyaltering the electrophoretic qualities of those molecules. I'll firstdiscuss preparing and running things i describe as a typical protein gel.This standard gel may be the system routinely employed for electrophoresis of mostprotein samples. I'll then discuss modifications that can be used for betterresolution of small proteins or peptides, as well as modifications for electrophoresisof nucleic acidity-protein fusions.
1. Standard Protein-Gel Electrophoresis
Materials 30% Acrylamide stock (37.5:1 acrylamide:bisacrylamide) Tris Base and Muriatic Acidity SDS Ammonium Persulfate TEMED Glycine EDTA 50% Glycerol b -Mercaptoethanol Bromophenol Blue Pre-Stained Molecular Weight Markers (optional)offered by GIBCO-BRL
PREPARING A PROTEIN GEL: Threebasic reagents are needed that may be prepared in the listing of materialsabove: (1) acrylamide gel buffers, (2) electrophoresis buffer, and (3)sample loading buffer.
The initial step would be to prepare the acrylamidegel. I more often than not prepare and run protein gels using 20 cm length glassplates, and can from time to time run them using theHoffer small-gel system.Standard protein gels are usually made up of two layers (Figure 1).The very best-most layer is called the stacking gel, also it comprisesabout 10-20% from the gel height. The stacking layer includes a low percentageof acylamide, typically 3.5-4.%, and it is buffered at pH 6.8. The lowerlayer of acrylamide, which comprises the other gel,may be the separating or resolving gel. The acrylamide power of theseparating gel varies based on the samples to become run. Generally, valuesof 8-15% acrylamide are utilized. The pH from the separating gel is 8.8. Thedifference in pH and acrylamide concentration in the stacking and separatinggel interface operates to compress the sample in the interface and providesbetter resolution and sharper bands within the separating gel. Some peoplechoose to omit the stacking gel, however personally don't suggest this.
The proteingel is ready inside a manner much like nucleic acidity polyacrylamidegels, however, if flowing a gel which contains a stacking gel, a bottomspacer can be used, and also the gel is put within the vertical position. Theacrylamide solutions are ready utilizing a stock of 30% acrylamide (37.5:1acrylamide: bisacrylamide) diluted towards the appropriate concentration in1X stacking/separating gel buffer. To supply a fine surface and interfaceat the top separating gel, isopropanol is positioned over the gel whileit polymerizes. Following the gel is totally polymerized, the isopropanolis put off, and the top gel is rinsed with deionized water. Ithen use Whatman paper to softly take in any excess water from boththe gel surface and glass plates. Using the comb in place in the topof the glass plates I pour the stacking gel on the top from the separating gel.This really is most easily made by pipeting the acrylamide solution in to the gel.
Establishing A PROTEIN GEL: Theprimary note to create here's that additionally to rinsing the wells, asis accomplished for nucleic acidity gels, the area at the end from the gel thatis produced by taking out the bottom spacer should be rinsed having a syringeneedle to purge any air bubbles out that will hinder electrophoresis.For this function, I've got a syringe needle which i have bent in order that it canreach up in to the crevice. Your final important note would be that the gel runningbuffer is really a tris-glycine buffer that differs from the buffer usedto prepare the gel. It's used in a 1X concentration.
RUNNING SAMPLES On The PROTEIN GEL: As you might expect, running samples on the protein gel is extremely similar torunning samples on the nucleic acidity gel. The sample is ready by dilutingit right into a concentrated sample loading buffer so the loading bufferis in a final power of 1X. The samples will be heated at 90 Cfor 3 minutes after which loaded to the gel, which isn't pre-run priorto sample loading. Protein gels are run more gradually than nucleic acidity gels,and therefore may need additional time. A gel 20 cm long generallyruns for around 3-4 hrs. Time needed is obviously variable dependingon the character from the sample. The samples are encounter the stacking gelat a couple of-3 watts for half an hour approximately, then the ability is increasedto 8-10 watts until completion. I usually include around the gel a lane thatcontains pre-stained molecular weight markers, which serve two purposes:(1) they permit you to monitor the gel, supplying an indication of methods thegel is running, and (2) provide molecular weight indicators to ensure that youcan figure out how lengthy to operate the gel.
FIXING AND DRYING PROTEIN GELS: When completed of electrophoresis the gel is separated just like a nucleicacid gel. The stacking gel is taken away, however, make sure to check it forradioactivity, and dump it accordingly. When the gel contains 32 Plabeled samples it may be dried directly while you would a nucleic acidity gel.When the gel contains 35 S-methionine samples the gel should befixed after which washed inside a patented solution known as "amplify", availablefrom Amersham, that will considerably shorten your exposure time for you to autoradiographyfilm. To repair the gel it's washed for 25 minutes inside a solution of: 50%methanol, 10% acetic acidity, and 40% water. The gel is rinsed in water, andthen washed within the "amplify" solution for 25 minutes, and isagain rinsed with water. The Amplify solution could be saved and reused 3-4times, or even more if there's very little free methione that diffuses intothe solution. With repeated use, this free methionine may cause an increasein the backgound degree of your gel. The gel is used in saran wrapand then to Whatman paper, and lastly it's dried under vacuum. Gels thatcontain 32 P samples are uncovered while engrossed in saran wrap.However, the saran wrap should be taken off gels which contain 35 Ssamples. It is crucial to use a skinny covering of baby powder tothe the surface of these gels so the autoradiography film doesn't adhereto the gel. I put it on, and wipe associated with a excess, having a kimwipe. Non-radioactivegels are fixed and stained with either coomassie blue, or silver stains.These protocols will vary, consider we don't presently run thesetypes of gels I won't cover them. If anybody ever finds themselves needingto stain a non-radioactive gel I'd gladly give a protocol atthat time.
1. Stacking Gel Buffer
125 mM Tris-HCl pH 6.8
.1% SDS
To create a 4X Stock (500 ml):
30.35 g Tris base pH'd to six.8 with HCl.
20. ml 10% SDS (or 2. g solid)
To create a 10X Stock (500 ml)
60.56 g Tris base
89.60 g Tricine
5. g SDS
400 ml ddH2 O
pH = 8.25 without adjustment
3. 8.five percent Separating Gel (30 ml):
8.5 ml 30% Acrylamide (37.5:1)
10. ml 3X Gel Buffer
8.5 ml 50% Glycerol
3. ml ddH2 O
4. 4% Stacking Gel (7.5 ml):
1. ml 30% Acrylamide
2.5 ml 3X Gel Buffer
2. ml 50% Glycerol
2. ml ddH2 O
RUNNING THE GEL: The gel isrun as described above. I run 2 ul of the 12.5 ul translation reaction onthe gel ( plus 4 ul of gel loading buffer). I have started to include .1%Triton X-100 and 2M Urea within my sample loading buffer since i appear tohave less things stuck towards the top of the separating gel. I generally runthe sample in to the stacking gel at 2 watts for 25 min. adopted for 20minutes at 6 watts. Then i boost the capacity to 8 watts and run the gelfor about 3 hrs. The bromophenol blue need not go to the underside.I love to run it about 60-75% towards the bottom.
FIXING AND DRYING THE GEL: Thesame as described above. A weekend exposure from the gel to autoradiographyis sufficient. Contact with the phosphorimager requires in regards to a 2-day exposure.
3. TBE-Urea gels: Electrophoresisof Nucleic Acidity-Protein Fusions.
STRATEGY: TBE-urea gels (nucleicacid gels) can be used as samples which contain nucleic-acidity-protein fusionsto give excellent resolution of protein and non-protein conjugates. Inthis system the negative control of individuals molecules that go into the gel isprovided through the attached nucleic acidity. For this kind of gel the sampleloading buffer used may be the standard urea loading buffer employed for nucleicacids. This technique is most effective when the sample isn't a complex mixture ofmany proteins that may form high molecular weight aggregates. Therefore,I haven't found results perfectly with crude translation reaction samples.Lately though, I've had moderate success with the addition of .1% triton X-100to both gel buffer and sample loading buffer. Nevertheless, I currentlydo not use this kind of gel for analyzing fusion samples, but instead theSDS-tricine system discussed above. I generally use "protein-gel acrylamide"stocks to organize these gels since the greater acrylamide:bisacrylamideratio (37.5:1) may even work better using these samples. I prepare the acrylamidesolution to contain 8 M urea, 1X TBE, and also the appropriate concentrationof acrylamide. I've generally run gels varying from 6-10% acrylamide.The present pool generates fusions which are about 94 KD,
87 KD nucleicacid, and seven.4 KD peptide. Of these molecules I'd use around a six percentcarbamide peroxide gel. I've run many gels of the type, and that i would gladly discussdetails and personalization for any specific application.
