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Variants of Gel Electrophoresis

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Variants of Gel Electrophoresis
Hello everybody this is Doctor Vishal Trivedi from department of biosciences and bioengineering IIT, Guwahati. And what we were discussing about the horizontal gel electrophoresis in our previous lecture. We have discuss about the experimental condition and how to perform the gel electrophoresis. We have also shown you a small demo clips how to do it in the lab and I hope that these are these 2 things would have been explained you the horizontal gel electrophoresis in a more detail and you will be able to perform the agarose gel electrophoresis in your own laboratory. So let us move on to discuss the different variants of the gel electrophoresis which you can do or which have been available.
So there are the SDS page which is actually a de-naturating page so as you know that theelectrophoretic mobility is actually been governed by charge by mass ratio. So if I keep the charge by mass ratio so if I keep the charge constant then the electrophoretic mobility is going tobe inversely proportional to the mass and that is what the condition for SDS page. So if I add theSDS into the gel electrophoresis the SDS is going to bind to the protein molecule.And as a result is actually going to give the negative charge and since the charge is imparted bythe SDS is going to be uniform for every protein. The charge component is going to be nullifiedAs a result the 3-D structure of the protein is destroyed and it migrates as per their subunitmolecular weight. age and if you run the same protein on to the native page and if youcalculate their electrophoretic mobility and if you use that information you can be able to answerthe question of the oligomeric status of the particular protein because the electrophoreticmobility in the SDS is going to be as per the molecular weight.Whereas, the electrophoretic mobility; in the native page is going to be as per the charge by massratio. Because if the protein is dimeric the mass if going to be of corresponding to the dimerwhereas in the SDS page it is going to be monomeric. So the combination of the SDS page andthe native page is going to give you the answer about the oligomeric status of the protein.Number 1 number 2 with the help of this native page you can be able to because the native pageis going to maintain the 3 dimensional confirmations.It is also going to maintain the activity of the protein so you can be able to do the functionalactivities you can be able to lot of activity assays when the protein is present in the gel and youcan answer you can resolve many questions related to the bio chemical activity of that particularenzyme or biochemical activity of that particular protein. Number 3 the native proteins becausethe native page is also going to allow you the 3 dimensional confirmations the native page can beused event for the studying the interaction between the 2 proteins.For example protein A and protein B is their protein A and B if they interact with each othertheir resultant charge resultant molecular mass is going to be very high. So that is how you canbe able to ask that particular question if you load protein A, protein B and protein AB complexthen the electrophoretic mobility is going to be different for the complex. Apart from that if you
see what the protein size and if you see the what kind of gel electrophoresis or what kind of thegel you have to use for seeing the better resolution and separation.But you see is that if you have the very small molecular weight for example the 4 to 40 kDaprotein then you can be able to use the 20% gel electrophoresis which means you have a if youhave prepared a 30% acrylamide solution actually contains 1% cross linking agent the (())(08:36) then from their you can be able to use the 20% gel, if you have if you are working withthe protein which are in the range of 4 to 40 kDa.But if you are working in the range of 12 to 45 kDa you can use 15% if you go to 10 to 70 youcan use the 12.5. If you use 15 to 100 you can use 10% and if you are working in the range of 25to 200 you can be able to use the 8% which means as the molecular weight is increasing you areactually decreasing the acrylamide. Because I think if you remember I have shown you that whenthe polyacrylamide is when the acrylamide is getting cross link by the bisacrylamidand thatactually creates a pore within the because of fibers are getting connected by the bisacrylamidIt actually creates a pore or the mess and from that mess the molecule as to pass through so ifyou take the very high concentrated acrylamide the high molecular weight proteins are not goingto enter. So for the practical references point of you this values are been utilized so that is bysimply by people has done the different types of experiment and that is how they come up withthis value.But the condition comes when you have to resolve a protein of 500 kDa for example or even2000 kDa protein. For example if you have such a large proteins or if you have the multi-mericprotein complex is how you are going to resolve that because if you drop this consultation tosuppose 5% okay or even to like 3% this lower consultation of the acrylamide is so less that itwill not go into give you the gel like structures it actually going to make the it is not actuallyenough to give you a gel which can be manipulated in a very simple way.So to solve this problem where you have a very large protein and protein complexes the peoplehave develop the new gel electrophoresis technique here they are using the multiple gels and thatis how they are been able to utilize and resolve this high molecular weight proteins. Apart fromthat suppose you have the protein of lower to this consideration. For example if you have the
amino acids which are you are interested to resolve on to the acrylamide gel then you cannot gobeyond the 20%. Because that is the maximum what you can prepare from the 30% acrylamidesolutions.But what you can see is that the 30% acrylamide solution as only the 1% bisacrylamide so if youhave a proteins of a very small size then what you can do is you can still be able to run the 20%gel but you can increase the percentage of the bisacrylamide. These kind of the gel are called asthe highly cross linked gels okay. So if you have a very small molecule weight you can use thehighly cross link gels where you can actually use the 2% or 3% bisacrylamide.Whereas if you have a very high molecule weight protein then you can you have to use acombination of different gels which we are going to discuss in a subsequent slide.Now you boil and let the acrylamide to be get dissolved the only thing is that you should not addthe temed and you should not add the APS at the system. So you prepare the completeSo for this purpose what you have to do is you have to rotate this cassette in thisdirection first okay. So you can imagine that we have just rotated this cassette by the 90 degreeand now you started filling from this side okay.So first you fill this one then you fill this one so when you rotate you have to block from the topas well and then you start pouring from the top and first you fill this one like that okay. So then
once you are done with the casting of all the up to the end then you can rotate it back again andthen you can start pouring the stacking gel, and you can be able to cast the you can put the comband you can cast the different wells, and then you just load the proteins which are already beenincubated with the different amount of urea.And as a result what you are going to see in the observation you are going to see it like forexample you have a 0 molar, 3 molar, 6 molar and 8 molar. So in the 0 molar you are going tosee a single band which is actually going to be corresponding to the tetramer. So I assume thatwe are dissolving a tetrameric protein. Now once it reaches to the 3 your tetramer considerationof the tetramer is going to be broken down.And then you are actually going to see some amount of trimer some amount of dimer and someamount of monomer which means this is actually going to be trimer this is going to be dimer andthis is going to be monomer. If you go further up then you band and concentration of thetetramer is going to be reduced whereas the all the other proteins are going to be increased. Butonce you reach to the 8 molar all these will get reach to the mole-meric proteins.So all will get broken down in to the monomer so if you can actually be able to follow these kindof study and if you do this kind of a experiments that actually will be going to tell you thestability of these individual oligomers towards the urea. So it will actually tell you that whichmonomer is going to be broken down and at what step the protein is losing its 3 dimensionalstructures and at what step it is actually losing its interaction with the neighbors.So, that the tetrameric protein is getting converted into trimeric and the dimeric and monomeric.And ultimately everything will get converted into the monomeric so at this stage all your proteingot unfolded whereas this stage all your protein is under the folding stage.
The 2 dimensional gels electrophoresis is a combination of isoelectric focusing followed by theSDS page in a perpendicular direction. Isoelectric points, separates the samples based on theirisoelectric pH it is indirectly related to the charge which is present on the proteins.to give you the 2 parts. You can imagine if I put and resolve the same example either using theisoelectric point or to the molecular weight then the samples will not get resolved properly orwill not get resolved completely.In general the analysis of complex bacterial lysate or tissue extract can produce even to 1000 to2500 well separated spots. With a sensitivity detection tool and image analysis softwareindividual of these spots can be identified under the different conditions. So the 2D gelelectrophoresis is very popular in terms of looking for the changes in the pattern of the proteinswhen you are treating a particular organism or bacteria with something which is actuallychanging the protieum of that particular organism.And because these changes are very subtle and these changes are very difficult to map simply byusing the property either the isoelectric point or to the molecular weight that is why people areusing the 2 dimensional gel electrophoresis to resolve them. And in general you are going toproduce 1000 to 2500 parts only and that actually is going to give you the enough separation tosee each and every spot.And once you got this spots you can be able to extract the protein from those parts and you canbe able to identify those spots and you can be able to identify the proteins. How to perform the 2dimensional; electrophoresis?(Refer Slide Time: 25:10)
The material what you require is for the 2 dimensional gel electrophoresis you require theisoelectric focusing strips. These isoelectric focusing strips are nothing but these strips of thecellulous where you have the ampholytes are arranged on to the strips of different charges. Sothis empollights are nothing but the amphoteric molecules and these strips are coated with thesemolecules so this region is actually going to be corresponding to a particular type of PI.So because of that when the proteins are going to run and when they reach to their isoelectricpoint they will get immobilized to that particular region. And because of that it they will getseparated based on the isoelectric point. Then you need a reagent for the SDS page and then youalso required the reagent because you want to do a sensitive detection so you can also require areagent for the silver staining.Then you require a trypsin because once you got the (()) (26:15) you have to (()) (26:16) them sothat the proteins are going to produce the peptides and then these peptides can be analyze in themass spectrometric to know what is the mass of this peptides and then you will be able toidentify the protein.(Refer Slide Time: 26:33)
This is as the multiple steps for example the step 1 you are going to do the protein extractions. Inthe protein extraction the tissue or the cell wall frozen into and will make a find powder then thissame powder is mixed with the chilled 10% Tca in acetone with 1% DTT. The tissue suspension
was incubated with 1 hour’s at 20 degree and the mixture is centrifuged at 35000g for 15 minutesat 4 degree.You can discard the supernatant and carefully dissolve the palate into the ice and acetonecontaining 1% DTT. Incubate the suspension and by going through this procedure ultimatelywhat you are going to get the material and before you load these material into the isoelectricfocusing strip you have to estimate the protein with the help of the either Lowry or the Bradfordmethod.So that it should not that to be you load very high quantity of the protein or very less quantity ofprotein. Because if you load very high quantity of the protein then the protein is going to overlapwith each other and operation is going to be compromise if you load very little then you are maymiss some of those protein which was present in the sample. But since the level was so low itmay not be it may not be up to the level of detections.(Refer Slide Time: 27:59)
Then the step 2 you are going to do the first dimensions so the IPG strips in this case we have Iam taking an example pH 3 to 10. So IPG strips either said you know are actually having theempollights which are coated and you can have a IPG strip of any range like 2 to 5, 2 to 10, 3 to10 is rehydrated overnight with 350 micro liter of 3 rehydration bar offers. And once it getrehydrated you can load the IPG strip with 1000 micro gram in a reswelling tray at roomtemperature.
This focused strips were equilibrated twice first equilibration in 50 milli meter trips we havecontaining strips and the second was perform solution containing 4% iodoacetamide instead ofDTT. So you are going to do the 2 focusing round in one of them it is going to have the DTT theother one you are going to have the iodoacetomide. Once the isoelectric focusing was conductedat 20 degree for running condition first hour at 500 volts followed by the 1000 volts for 2 hoursand finally the 16 hours at 3000 volts.So when you run these for the 16 hours at 300 volts the proteins are going to migrate throughoutthe isoelectric focusing strip and then it will get immobilized to its individual spots. The IPGstrips will be taken out from the operators and the second dimensional separation will beperformed in the SDS page in a vertical slab of the acrylamide.(Refer Slide Time: 29:37)
Now once you are done with this first dimensions then what you are going to have is you aregoing to have the IPG strips okay. And then what you have to do is you take the IPG strips andput it into the acrylamide solutions so what you are going to do? You are going to do first castethe resolving gels okay but you do not need to cast the stacking gels and so first you cast theresolving gel and you put the your strip okay and then you actually pour the stacking gel andbecause of that you are actually going to seal the differences between the IPG strip as well as theSDS page.
And then it becomes the continuous 1 gel and then you are going to perform the seconddimension SDS page as we were discussing about the vertical gel electrophoresis. The proteinswhich are actually going to be migrated like this are now going to run in these direction okaywhich means all these protein is going to be concentrate and that is how you are actually going toget a spot from this protein. So you might have multiple proteins which are been present in thisparticular spots and that is why you are going to see the multiple spots from a single IPG stripfocusing area.Which means at a PI of 3.1 you might have 5 protein and all these 5 proteins may have adifferent molecular weight. So that is why you are going to see the proteins of the spots of thedifferent positions and same will be true for multiple places. Once you got the spots you can beable to you know analyze these pot pattern on the second dimension gel and you can be able tocompare it with the untreated samples or the different treatment samples and that is how itactually going to tell you that what are the spots are differentially been expressed.Which means these are the spots which are additionally been present and that is the spots youcan be able to extract out from the gel and that you can be able to use the downstream 2dimensional gel electrophoresis approaches. And (()) (32:03) for example you can do thetrypsmization and then you do the multi mass and all that and you can be able to identify theproteins.There are couple of good MOOC courses are available from the IIT Bombay if you are interestedto study the 2 dimensional gel electrophoresis as well as the proteomic. So if you are interestedyou can actually go to those IIT Bombay’s and MOOC courses and you can study that in more indetails. So here we are not going to discuss each and every those steps I am just trying to tell youthat these are the actually obstance which are available for you to utilize gel electrophoresis toanswer many critical questions.So in typical what you have what we have done so what we have discussed so far that you arefirst going to take the cell or the tissue, you are going to prepare the extract the first you aregoing to run this extract on to the IPG strips. And that will be going to resolve them as in the
form of the bands on to these strips, and then what you are going to do is you are going to loadthese strip on to the SDS page.So each strip, each spot is or each band it now going to be resolve into the individual spots andthat is how you are going to get the 1000 to 2500 different spots and these spots can be cut outfrom the gel and can be done for the downstream applications like the proteous treatment themass spectrometry.(Refer Slide Time: 33:44)
Now we have discuss about the native page we have discuss about the SDS page we have discussabout the agarose and acrylamide gels and we have also discuss about the 2 dimensional gelelectrophoresis. What is the limitation of the native page is that it always governed by theintrinsic charge of the particular protein which means if the protein is negatively charge it isactually going to run as per the negative charge.But you know that the electrophoresis operates has a negative electrode on to the top and positiveelectrode at the bottom. But what will be the condition if you have a mixture where you aregoing to have the positively charged and as well as the negatively charged protein. In those kindof complex mixture you will not be able to utilize the native page because either the positivelycharged protein will run or the natively charged protein runs.
Irrespective of whether so if you have a complex mixture where you have the positively chargedor the negatively charged proteins you have to run the horizontal page. So horizontal page issimilar to the agarose gel but you are using the acrylamide instead of the Agarose.(Refer Slide Time: 35:11)
So, horizontal gel electrophoresis in this apparatus the complex biological sample is resolved asper their charge and move to the counter charge electrode. Which means the positive will movetowards the negative and the negative will move towards the positive. The sample; loaded in themiddle of the gel get resolved based on their mass by charge ratio. The gel cassette is designed toprepare agarose gel is not appropriate to cast the polyacrylamide gel due to the exposer of gelwith the environmental oxygen.So the gel cassette what we use for the agarose gel electrophoresis is not good enough to cast thepolyacrylamide gels. Because, it is been open from the top so because of that it is actually goingto get the direct entry of oxygen; and you know that the oxygen is a exhibitor of the acrylamidepolymerization. So if the oxygen is present the polyacrylamide is not going to get polymerized togive you the page.So because of that you require a specialized native page operator which actually can resolve thesample based on the charge by mass ratio. The horizontal native page separates the proteinmixture with the high resolution and the protein migration is corresponding well with the massby charge ratio.
(Refer Slide Time: 36:34)
So first discuss about the instrumentation part so the design the gel cassettes. The gel cassetteconsists of 3 plates the 1 big plate which is the plate number 1 and the 2 small plates which arenumber 2 and 3 are 2 mm thick glass slide is stick to the large plate to give the build spaceswhich means you are actually going to put the 2mm thick glass slide on to the slide. And thatactually is going to give you the spacers the gel cassette is sealed with the thick foam on to theboth sideOkay on the both side you have a very thick foam which is actually going to use for the sealingof this particular operators which is impregnated with agarose to avoid the leakage. Then the gelcassette is assembled with the help of a binder clips with a 1mm 1 centimeter gap to place thecomb. Which means you have this main plate you have a comb you have a foam on this side youhave spacer on both the sides.And then you what you are going to do is you are going to place the number 2 you are going toplace the number 3 like this and then you are going to put the clips on both the sides. So that thiswhole operator is going to be assembled as 1 and then you are going to start casting thepolyacrylamide gels.(Refer Slide Time: 38:04)
The casting of the horizontal native acrylamide gels the gel cassette is assembled by the binderclips to keep 1 centimeter gap between them to place the combs. The leakage of the cassette waschecked by the water before pouring the acrylamide solution. So what you have is you have amain plate where you are going to have the foam at the bottom and the top so first what you do isyou are so this is the plate number 1.The first what you do is put the plate number 2 is okay put the clips on both the sides and thenyou pour the liquid okay. And once the it is get resolved and it get polymerized then you have torotate this okay and then you put t number 3 then this side and you bring it down okay and thenpour it again. So you can imagine that this is like this if you are going to see for this so number 2will go on top and number 3 will come at the bottom and that is how you are going to pour intothis.So actually the casting is going to be a 2 step event first you cast for the number 2 then you castfor the number 3 and then the middle portion is going to be empty and the thin layer of waterequilibrated brutanol is over-layed on top of resolving gel. Same procedure is adopted to cast theresolving gel on the other side of the glass plate. Gel cassette is placed horizontally and stackinggel is poured and a comb is placed to cast the wells which means once these are things done thenyou take the plate like this okay.
So this side is casted this side is casted then you pour the stacking gel in between and put acomb. So if you put the comb the well is going to be prepared just like it was prepared for theDNA.(Refer Slide Time: 40:04)
Now running of the gel the sample preparation the protein samples are mixed with the 5x loadingdye containing 40% sucrose 10% of bromophenol blue and 10% methylene blue. Bromophenolblue is a anionic dye and used to monitor the mobility of proteins on the anodic side whereas MBis a cationic dye to track the moment of the other side. The electrophoresis once you put thatonce the stacking gel is polymerized the comb binder clips and foam pads are removed and wellsare washed with water and 1x the native Tris-glycine running buffer.The gel cassette is placed in the horizontal direction in the electrode chamber. The chamber isfilled with the chilled 1x native Tris-glycine running buffer to the level just enough to touch tothe glass plate which means if you have this and you have kept the glass plate like this okay. Sothis side you have a chamber you connect these with a something so that it actually take up thebuffer but it is not a continuous mode of running.It is going to be discontinuous mode so that the current goes through the gel from 1 bufferchamber to the next buffer chamber. So the principle remains the same except that now we aredoing the electrophoresis in the horizontal directions. Load the samples up to the 20 mica liter anelectrophoresis is performed with the constant 100 volts in a cold room. Because the
electrophoresis is going to be for 18 to 20 hours so it as to be in the cold room so that the sampledoes not get denatured and destroyed.(Refer Slide Time: 41:51)
Then once you are done with that you do the staining and de-staining after the electrophoresis isover gel is removed from the cassette with the help of a scalpel and the stained with thecoomassie brilliant blue. The whole process of staining and de-staining of the gel complete isless than 3 hours and what you can is that i have loaded a bacterial lycate and what you can see isthat the positively charged proteins are going towards the negative electrodes and the negativelycharged proteins are migrating towards the positive electrodes and that is how you can be able to.And all the both of these samples are native in so that is how you can be able to resolve and youcan be able to visualize the pattern of the positive as well as the negative protein present in thesame mixture using the horizontal gel electrophoresis.(Refer Slide Time: 42:45)
What are the advantages of this gel electrophoresis the horizontal gel electrophoresis can be usedin this conjugation with SDS page to separate and analyze the complex biological samples. It isuser friendly and no specialized equipment is required the native page native preparative gel ispurify in bulk quantity the protein in the bulk quantity for the antibody development as well asfor the activity as said.Moreover this design does not require any specialized fabrication and in allow user to caststacking and resolving gel together. Which means the horizontal page is giving you the manyadvantages where you can actually utilize them to resolve the proteins of the positively as well asthe negatively charged which are present in the sample together. Number 2 because it is actuallyresolving the sample based on the charge by mass ratio you can be able to utilize the gel inconjugation with the SDS page, and that actually will allow you to resolve the very complexbiological samples.And there are other advantage that is user friendly compare to the other TDS the horizontal page(()) (43:59) are available and so with this we would like to conclude our lecture here. In oursubsequent lecture we are actually going to discuss the experiments as well as the researchproblems what are been related to the electrophoresis. And we also going to discuss these themost staining techniques which are also available in the which are also been used in theelectrophoresis.
So with this I would like to conclude my lecture here in a subsequent lecture we will be going todiscuss the more topics related to the electrophoresis thank you.