BIo Lab Qs- SDS-PAGE, Microarray

[Ocean5]

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I've been trying to learn DSD- page and Microarray experiment (whcih by the way could be used for many things) from Wikipedia, but wiki gives too much detail that overshadows the important stuff sometime. Since we didn't do these experiments in our bio lab, I need a core explanation of the experiment. Could some one please explain these experiments. Thank you.

 

[TheWiredNerv]

SDS page makes it so macromolecules are only separated by mass in electrophoresis.

 

[Ocean5]

SDS page makes it so macromolecules are only separated by mass in electrophoresis.

I'm sorry ! I didn't get it. SDS-Page make it? what do you mean by it?

 

[sacjumpman]

I'm sorry ! I didn't get it. SDS-Page make it? what do you mean by it?

http://sps.k12.ar.us/massengale/ap_powerpoints_7th.htm

On that page, read Chapter 20. It is basically campbell's bio summed up rather well.

SDS-PAGE: separates proteins based on size. SDS is sodium dodecyl sulfate. This breaks up the proteins interactions such that it has an overall negative charge. It will now move solely on the basis of size, rather than charge.

It moves to the anode since it is an "anion" - negatively charged. The anode is positively charged.

This contrasts with Isoelectric focusing which will separate a protein based on its charge. There is a recent post by jigabodo that sums up Isoelectric focusing really well.

 

[sacjumpman]

Also, don't sweat microarray too much.

I have yet to hear about it coming up at all, not to say it won't, but its probably not a high, high priority.

But understand your different gel electrophoresis and FRAP, those are the infamous ones this summer.

 

[jigabodo]

When proteins are in their funtional state, they each possess unique 3D globular structures so it would be difficult to measure their MW.

First off, SDS PAGE stands for sodium dodecyl sulfate polyacrilamide gel eletrophoresis (can't spell...) The gel part of SDS PAGE are actually composed of many pores, which allow proteins of different sizes to travel at different rate. (Think of this as like a road full of road kills/trash everywhere. If you are a motorcycle it would be easier for you to manuver pass these things therefore travel faster whereas if you are a huge truck, you are doomed to hit something so you will travel slower.)

However, the gel alone cannot work to separate proteins based on MW. Why, because different proteins will have different shapes, so if 2 proteins have similar MW yet different structures, they will migrate at different rates.

This is when SDS comes in handy. Presence of SDS will denature all proteins to primary structures...so essentially with SDS they will all look the same, aka the random coil structure - now you may separate proteins solely based on MW because they all have the same structures.

SDS also adds negative charge to proteins, and this is also important for separation. If the protein was originally uncharged, it will not move at all under electric current. However, because now negative charges are being added due to SDS, protein willl now be able to migrate to anode based on respective MW.

Now, you may wonder, wouldn't the different charges of SDS on protein contribute their migration rate in addition to MW? It does not, because SDS actually binds to proteins on a constant MASS TO CHARGE ratio, so essentially the proteins under SDS will be separated solely based on MW.

hope it helps. This might not be very good, please feel free to ask if got more questions.

 

[sacjumpman]

That's great, man. Thank you.

For native electrophoresis, we now separate based on the 3-D shape of the protein, i.e. it doesn't become denatured?

Does native electrophoresis have any other name we should be familiar with?

 

[Ocean5]

When proteins are in their funtional state, they each possess unique 3D globular structures so it would be difficult to measure their MW.

First off, SDS PAGE stands for sodium dodecyl sulfate polyacrilamide gel eletrophoresis (can't spell...) The gel part of SDS PAGE are actually composed of many pores, which allow proteins of different sizes to travel at different rate. (Think of this as like a road full of road kills/trash everywhere. If you are a motorcycle it would be easier for you to manuver pass these things therefore travel faster whereas if you are a huge truck, you are doomed to hit something so you will travel slower.)

However, the gel alone cannot work to separate proteins based on MW. Why, because different proteins will have different shapes, so if 2 proteins have similar MW yet different structures, they will migrate at different rates.

This is when SDS comes in handy. Presence of SDS will denature all proteins to primary structures...so essentially with SDS they will all look the same, aka the random coil structure - now you may separate proteins solely based on MW because they all have the same structures.

SDS also adds negative charge to proteins, and this is also important for separation. If the protein was originally uncharged, it will not move at all under electric current. However, because now negative charges are being added due to SDS, protein willl now be able to migrate to anode based on respective MW.

Now, you may wonder, wouldn't the different charges of SDS on protein contribute their migration rate in addition to MW? It does not, because SDS actually binds to proteins on a constant MASS TO CHARGE ratio, so essentially the proteins under SDS will be separated solely based on MW.

hope it helps. This might not be very good, please feel free to ask if got more questions.

You're my hero!

 

[Ocean5]

http://sps.k12.ar.us/massengale/ap_powerpoints_7th.htm

On that page, read Chapter 20. It is basically campbell's bio summed up rather well.

SDS-PAGE: separates proteins based on size. SDS is sodium dodecyl sulfate. This breaks up the proteins interactions such that it has an overall negative charge. It will now move solely on the basis of size, rather than charge.

It moves to the anode since it is an "anion" - negatively charged. The anode is positively charged.

This contrasts with Isoelectric focusing which will separate a protein based on its charge. There is a recent post by jigabodo that sums up Isoelectric focusing really well.

Thank you. This site is good!

 

[jigabodo]

That's great, man. Thank you.

For native electrophoresis, we now separate based on the 3-D shape of the protein, i.e. it doesn't become denatured?

Does native electrophoresis have any other name we should be familiar with?

Native page separates based on both 3D shape and MW, and the proteins do not become denatured. It is often used to analyzed shape of proteins and used together with SDS. e.g. If I know 2 proteins have the same MW based on SDS, then I will run it with native page to get more info about their shape.

I think it has another name, but I forgot....

Sorry

 

[sacjumpman]

Native page separates based on both 3D shape and MW, and the proteins do not become denatured. It is often used to analyzed shape of proteins and used together with SDS. e.g. If I know 2 proteins have the same MW based on SDS, then I will run it with native page to get more info about their shape.

I think it has another name, but I forgot....

Sorry

It's all good. You've been more than helpful with all this. You definitely deserved your scores, you know this stuff well.

 

[craptastic]

Native page separates based on both 3D shape and MW, and the proteins do not become denatured. It is often used to analyzed shape of proteins and used together with SDS. e.g. If I know 2 proteins have the same MW based on SDS, then I will run it with native page to get more info about their shape.

I think it has another name, but I forgot....

Sorry

Seriously thanks ALOT jigabodo. You've been a great help yet again. Feel free to enlighten us with more wisdom. 🙂

 

[jigabodo]

Bumping. Good luck on your DAT, guys.

 

[bumpski20]

I just skimmed this and i didnt see anything about microarray data so ignore this if theres already info. oh and the botton on the keyboard between c and b is mission on my laptop so im putting a * in its place.

microarray analysis allows you to study the gene expression of millions of genes all at once.

heres the process (one of many) of cloning the genes to the microarray: first extract the RNA. separate the mRNA from the other RNAs by passing it through beeds with oligoDT (not sure on the spelling of that but basically it has a poly T tail so the mRNA, which has poly A tail, will stick to it and the rest will pass through) (side note- this separation method is an example of affinity chromatography). once the mRNA is isolated you obtain cDNA (coding DNA) through re*erse transcriptase. from here you clone the cDNA to the mircroarray. this would be a cDNA microarray but there are others (like RNA, DNA, etc).

heres an example of what you can test: in our cellnmolec lab we put yeast through a diauxic shift. time point 0 was when the yeast were in a glucose rich en*ironment so they metabolize glucose to ethanol through fermentation. more and more glucose was taken away until there was no glucose and the yeast were forced to metabolize ethanol through aerobic respiration. the RNA that was extracted periodically throughout this diauxic shift so that it represtented the gene expression of yeast under glucose rich en*ironments, no glucose, and time points inbetween.

analysis: a microarray looks like a square with thousands of different colored circles lit up. if you ha*e the right computer program, it quantifies the results. you can choose which genes you want to analyze from here. one of the biggest points of this lab was that the data showed the expression of cytoplasmic ribosomes to be extremely high towards the begining (fermentation was at the begining and fermentation takes place in the cytoplasm...this makes sense!) and little to no expression in mitochondrial ribosomes (again, fermentation was at the begining. this isnt in the mitochondria, so little expression of these ribosomes also makes sense!). can you guess if the expression changed throughout the diauxic shift and how?? this makes sense as well...towards the end of the shift (when yeast were going through aerobic respiration which takes place in the mitochondria) mito ribosomes were highly expressed and cytoplasmic ribosomes were barely expressed.

of course you can analyze a lot more then ribosomes, but i think the example is the easiest to grasp. sorry if theres a lot of typos, im really tired...i just hope it makes sense!