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Another procedure, gel filtration Chromatography, in which we separate the proteins based on size. | Analysis of Protein Purification (Part II).txt |
And so following that, we extract that sample, we place it into this weld, and this is the band that we get. | Analysis of Protein Purification (Part II).txt |
And so this band disappears, other bands disappeared. | Analysis of Protein Purification (Part II).txt |
So what that means is we're slowly purifying the protein where we're moving those unwanted proteins as shown by padded increase in the band distribution number. | Analysis of Protein Purification (Part II).txt |
So that is what should be shown by this table. | Analysis of Protein Purification (Part II).txt |
So let's calculate this box. | Analysis of Protein Purification (Part II).txt |
In this box, as always, the specific activity is 85,000 divided by 100. | Analysis of Protein Purification (Part II).txt |
The two zeros cancel out and we have 850 divided by one and that gives us 850. | Analysis of Protein Purification (Part II).txt |
Now, what about this quantity? | Analysis of Protein Purification (Part II).txt |
So to calculate this, we simply take this divided by that. | Analysis of Protein Purification (Part II).txt |
So the specific activity of that mixture divided by the original mixture and we get 85. | Analysis of Protein Purification (Part II).txt |
So because this divided by this gives us 85. | Analysis of Protein Purification (Part II).txt |
And let's use purple for that. | Analysis of Protein Purification (Part II).txt |
Okay? | Analysis of Protein Purification (Part II).txt |
So what that means is this extracted mixture, following these processes up to this point is 85 times as pure as this initial mixture. | Analysis of Protein Purification (Part II).txt |
And finally, let's carry out the final one. | Analysis of Protein Purification (Part II).txt |
So in the final step, in step E, we take our extracted mixture of proteins and we expose it to affinity Chromatography. | Analysis of Protein Purification (Part II).txt |
And in affinity Chromatography, we separate our proteins based on their specific ability to bind to these special molecules. | Analysis of Protein Purification (Part II).txt |
And this usually creates a very high specific activity value because as we know, enzymes only bind to specific substrate to specific molecules. | Analysis of Protein Purification (Part II).txt |
So let's see if that's true by taking the extracted mixture and placing it into our last well. | Analysis of Protein Purification (Part II).txt |
So what we produce is a band distribution that only contains a single band. | Analysis of Protein Purification (Part II).txt |
And what that usually means is we have isolated that protein of interest because this band consists of a protein that has a specific type of mass, a specific type of size. | Analysis of Protein Purification (Part II).txt |
Now let's owe and by the way, let's calculate what the yield in this case was. | Analysis of Protein Purification (Part II).txt |
To calculate the yield, we follow this equation. | Analysis of Protein Purification (Part II).txt |
So this divided by this gives us 85 divided by 200, which is 42.5 divided by 100 multiplied by 100, that gives us a percent of 42.5. | Analysis of Protein Purification (Part II).txt |
Okay? | Analysis of Protein Purification (Part II).txt |
So let's go back to this step. | Analysis of Protein Purification (Part II).txt |
So this basically means that we have successfully isolated that protein. | Analysis of Protein Purification (Part II).txt |
We basically have this protein that consists of a single type of mass. | Analysis of Protein Purification (Part II).txt |
Now let's see that the specific activity increases and this increases. | Analysis of Protein Purification (Part II).txt |
And let's make sure that the yield didn't decrease by too much. | Analysis of Protein Purification (Part II).txt |
So if we have at least 30% here, that is a good procedure. | Analysis of Protein Purification (Part II).txt |
So let's take a look at E to calculate this. | Analysis of Protein Purification (Part II).txt |
We take this divided by two and we get 35,000. | Analysis of Protein Purification (Part II).txt |
So we see that in fact, this procedure has a high specific activity. | Analysis of Protein Purification (Part II).txt |
And that makes sense because affinity Chromatography separates these enzymes based on their ability to bind to specific enzymes. | Analysis of Protein Purification (Part II).txt |
And so in our mixture, when we run our mixture proteins along that column of that affinity chromatography setup, only the protein that we want to study will bind to our beads inside that column, the other proteins will essentially go down because enzymes only bind to specific types of proteins. | Analysis of Protein Purification (Part II).txt |
And so that's why this is such a high value, because usually affinity chromatography separates by a very, very large margin. | Analysis of Protein Purification (Part II).txt |
Now, what about the purification level? | Analysis of Protein Purification (Part II).txt |
So this divided by this gives us 3500. | Analysis of Protein Purification (Part II).txt |
And what that means is, after all the procedures that we conducted, the final extracted mixture is 3500 times as pure as that initial sample, and that is a high amount. | Analysis of Protein Purification (Part II).txt |
Now, for this set of procedures to actually be good, this yield has to be a high enough yield. | Analysis of Protein Purification (Part II).txt |
So let's see what that yield is. | Analysis of Protein Purification (Part II).txt |
So we take 70,000 divided by 200,000. | Analysis of Protein Purification (Part II).txt |
That gives us 70 divided by 200 or 35 divided by 100. | Analysis of Protein Purification (Part II).txt |
We multiply that ratio by 100 and we get 35%. | Analysis of Protein Purification (Part II).txt |
And this is a high enough yield. | Analysis of Protein Purification (Part II).txt |
So 35% is enough to basically mean we can work with that extracted sample, the protein, to basically study that protein in different ways. | Analysis of Protein Purification (Part II).txt |
So these are the five quantities that we have to calculate every time we carry out one of these procedures. | Analysis of Protein Purification (Part II).txt |
And then we also normally expose our extracted portion to SDS page to basically help us visualize if our technique is actually working. | Analysis of Protein Purification (Part II).txt |
And if we combine these two methods, that gives us a very good idea if the purification technique is working. | Analysis of Protein Purification (Part II).txt |
So normally, as we go from A to E, the number of bands should decrease. | Analysis of Protein Purification (Part II).txt |
And what that means is we're getting rid of those unwanted proteins and we're focusing in on that protein that we actually want to study. | Analysis of Protein Purification (Part II).txt |
And that is confirmed by these measurements because as we purify the sample, the specific activity has to increase and so should our purification level. | Analysis of Protein Purification (Part II).txt |
And we also have to keep track of the yield. | Analysis of Protein Purification (Part II).txt |
We don't want our yield to drop to a very small amount. | Analysis of Protein Purification (Part II).txt |
For example, if following our procedure, this yield would have been, let's say 5%, then this procedure would not have been efficient because at the end, even though we would have had a high purification value, that yield is not enough. | Analysis of Protein Purification (Part II).txt |
5% would not have been enough to basically create a mixture protein that is actually usable and workable in a laboratory setting. | Analysis of Protein Purification (Part II).txt |
So whenever we're purifying proteins, we have to make sure that the purification level is high and that yield is high enough for us to actually work with that final purified mixture of protein. | Analysis of Protein Purification (Part II).txt |
Although DNA molecules contain the genetic information that is needed to build proteins the DNA molecules themselves are not used directly in protein synthesis. | RNA Polymerase .txt |
And that's because we want to actually prevent damage to the DNA molecules. | RNA Polymerase .txt |
And so what we do is in a process known as transcription we synthesize RNA molecules from DNA molecules. | RNA Polymerase .txt |
And what these RNA molecules are they're copies of specific segments of the DNA molecule and these segments usually contain important genes. | RNA Polymerase .txt |
So before we can synthesize proteins RNA molecules must be synthesized from DNA molecules in a process known as transcription. | RNA Polymerase .txt |
And just like the process of DNA replication involves this protein known as DNA polymerase that catalyzes the formation of a phosphodiastor bond in the process of transcription. | RNA Polymerase .txt |
When we form the RNA from the DNA there is a molecule, a protein known as RNA polymerase that catalyzes the formation of the phosphodia Esther Bond. | RNA Polymerase .txt |
So RNA polymerase catalyzes the initiation and the elongation of that RNA polynucleotide chain. | RNA Polymerase .txt |
And to see what we mean by that, let's take a look at the following diagram. | RNA Polymerase .txt |
So in the diagram in this chemical equation we basically describe how this reaction takes place and what RNA polymerase does. | RNA Polymerase .txt |
So let's suppose we have an RNA chain and we're extending that RNA chain. | RNA Polymerase .txt |
So we're building our RNA molecule. | RNA Polymerase .txt |
Now, so far in our RNA molecule we have N number of nucleotides. | RNA Polymerase .txt |
And let's suppose we want to add one more nucleotide. | RNA Polymerase .txt |
We want to add one more ribonucleotide triphosphate. | RNA Polymerase .txt |
Now, what RNA polymerase does and we'll discuss this in much more detail in just a moment what RNA polymerase does is it essentially attaches the ribonucleocide triphosphate onto the RNA molecule to form an RNA molecule that is extended by one nucleotide. | RNA Polymerase .txt |
So here we have N number of ribonucleotide triphosphates but here we have N plus one. | RNA Polymerase .txt |
And in the process we build this phosphodiastor bond between the ribonucleotide triphosphate and the RNA chain and we release a single pyrophosphate molecule. | RNA Polymerase .txt |
So PP stands for a Pyrophosphate and in fact it's the breakdown of the Pyrophosphate that drives this transcription reaction forward as we'll discuss in much more detail when we'll discuss the process of transcription. | RNA Polymerase .txt |
So this is the general equation that describes RNA polymerase. | RNA Polymerase .txt |
Now, to actually work, what does RNA polymerase actually need? | RNA Polymerase .txt |
Well, it needs three different things. | RNA Polymerase .txt |
It basically means the building blocks, the ribonucleotide triphosphates and there are four different types. | RNA Polymerase .txt |
So we have adenosine five triphosphate we have guanosine five triphosphate we have citadine five prime triphosphate and we have uridine five prime triphosphate. | RNA Polymerase .txt |
And these building blocks are needed to actually synthesize and elongate that polynucleotide chain. | RNA Polymerase .txt |
Now, number two, it actually needs a preexisting DNA template molecule. | RNA Polymerase .txt |
So usually we're dealing with a double stranded DNA molecule and a double stranded DNA molecule works the best. | RNA Polymerase .txt |
But in some cases we can also use single stranded DNA molecules as templates. | RNA Polymerase .txt |
Now, why do we need a template molecule? | RNA Polymerase .txt |
Well, we need the DNA template to basically copy that genetic information. | RNA Polymerase .txt |
And it's the template, that DNA template that is used to basically bring those complementary bases to that elongated polynucleotide chain. | RNA Polymerase .txt |
So although single stranded DNA molecules work, double stranded DNA molecules are more effective and more common as templates. | RNA Polymerase .txt |
So RNA polymerase uses this DNA template to basically build a polynucleotide chain that contains complementary bases as we'll see in just a moment. | RNA Polymerase .txt |
And finally, inside the RNA polymerase molecule at the center there is a pocket that can fit a divalent metal atom. | RNA Polymerase .txt |
Now, what is a divalent metal atom? | RNA Polymerase .txt |
Well, it's a metal atom that can gain a positive charge, a charge of positive two. | RNA Polymerase .txt |
So he can lose two electrons and gain a positive charge of two. | RNA Polymerase .txt |
And two types of atoms that work in this particular case are magnesium so, Mg and Manganese MN. | RNA Polymerase .txt |
And the reason we need this metal atom is because it acts as a cofactor, it increases the efficiency of this enzymes activity. | RNA Polymerase .txt |
Now let's take a look at the following diagram that basically describes in detail how that bond is formed. | RNA Polymerase .txt |
And notice this phosphodiasta bond is formed in a very similar way to how the phosphodiesta bond is formed when DNA polymerase replicates that DNA molecule. | RNA Polymerase .txt |