Any Unusual Bio material or Lab Concepts on DAT?

[CuddlesUCLAbear]

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Hi everyone,

Has anyone ran into or have heard of any surprising questions on the DAT? Specifically any questions that were on the DAT which was not covered by the typical study tools (kaplan, schaum's, etc.)?

Thank you all! = )

 

[jigabodo]

-Photobleaching
-FRAP/FLIP
-SDS PAGE
-IEF
-Agarose gel Electrophoresis
-Elution of different compounds
-Practical applications regarding separation of proteins
-Practical applications regarding identity of proteins

Thats all I can think of right now...

 

[craptastic]

What are:

-Elution of different compounds
-Practical applications regarding separation of proteins
-Practical applications regarding identity of proteins

Could you please explain the extent we need to know about these? thanks.

 

[jigabodo]

IEF = isoelectric focusing. It is a method of choice to separate proteins/oligopeptides based on their pI. In short, you will make a gel with electrical current, where one side is anode and one side is cathode. You will also prepare a pH gradient on the gel so that you will be able to check for the protein of choice's pI. You will then put the proteins/oligo into the gel, and see how it migrates accordingly.

For example, lets say your protein has a pI of 3, and you begin by putting it into the middle of the gel, which correspond to pH 7. Because at the middle of the gel (pH 7) the solution is more basic than the protein, it will strip H+ off the protein and thus make it have a negative charge. The protein will then start migrating to the anode because anode attracts negatively charged particles. The process will occur until the protein of choice reaches pH of 3 at the gel. At this point, the protein will no longer have a charge because pH=pI, and therefore unaffected by the electrical current, and now the migration will cease.

Here is an graphical illustration of IEF: http://fig.cox.miami.edu/~cmallery/255/255tech/focusingp555.jpg

 

[jigabodo]

What are:

-Elution of different compounds
-Practical applications regarding separation of proteins
-Practical applications regarding identity of proteins

Could you please explain the extent we need to know about these? thanks.

Sorry, didnt see the other 2.

Questions regarding separation of proteins are actually quite straight forward, you just have to know the techniques and what is the basis of separation. 2 good ones to know will be differential centrifugation and density centrifugation. For example, if I give 2 proteins, one got MW of 900 and density of 2 g/cm3 and the other has MW of 650 and density of 2 g/cm3, which technique would you use?

Questions regarding identities of proteins are trickier, but its def. worth to learn it because it actually showws up on my DAT. For example, lets say you got a cell, lyzed it open and did SDS PAGE and commassie blue stained it and got 3 distinct bands. You then got another cell, from the same strain and then treat it with protease. After protease worked for a while, you added protease inhibitor AND THEN lyzed then cell open. You then ran it on SDS PAGE + commassie staining but now you only found 2 bands. Based on the information, how many of the 3 are internal/integral proteins?

 

[Shermanmurman]

IEF = isoelectric focusing. It is a method of choice to separate proteins/oligopeptides based on their pI. In short, you will make a gel with electrical current, where one side is anode and one side is cathode. You will also prepare a pH gradient on the gel so that you will be able to check for the protein of choice's pI. You will then put the proteins/oligo into the gel, and see how it migrates accordingly.

For example, lets say your protein has a pI of 3, and you begin by putting it into the middle of the gel, which correspond to pH 7. Because at the middle of the gel (pH 7) the solution is more basic than the protein, it will strip H+ off the protein and thus make it have a negative charge. The protein will then start migrating to the anode because anode attracts negatively charged particles. The process will occur until the protein of choice reaches pH of 3 at the gel. At this point, the protein will no longer have a charge because pH=pI, and therefore unaffected by the electrical current, and now the migration will cease.

Here is an graphical illustration of IEF: http://fig.cox.miami.edu/~cmallery/255/255tech/focusingp555.jpg

that was really helpful, thanks👍

 

[pbure]

Sorry, didnt see the other 2.

Questions regarding separation of proteins are actually quite straight forward, you just have to know the techniques and what is the basis of separation. 2 good ones to know will be differential centrifugation and density centrifugation. For example, if I give 2 proteins, one got MW of 900 and density of 2 g/cm3 and the other has MW of 650 and density of 2 g/cm3, which technique would you use?

Questions regarding identities of proteins are trickier, but its def. worth to learn it because it actually showws up on my DAT. For example, lets say you got a cell, lyzed it open and did SDS PAGE and commassie blue stained it and got 3 distinct bands. You then got another cell, from the same strain and then treat it with protease. After protease worked for a while, you added protease inhibitor AND THEN lyzed then cell open. You then ran it on SDS PAGE + commassie staining but now you only found 2 bands. Based on the information, how many of the 3 are internal/integral proteins?

o man..i dont get any of that. how could they formulate that as a multiple choice question? i feel like i'd have to write an essay on it or something

 

[craptastic]

aww man THANKS ALOT jigabodo that was really helpful!

 

[craptastic]

Questions regarding identities of proteins are trickier, but its def. worth to learn it because it actually showws up on my DAT. For example, lets say you got a cell, lyzed it open and did SDS PAGE and commassie blue stained it and got 3 distinct bands. You then got another cell, from the same strain and then treat it with protease. After protease worked for a while, you added protease inhibitor AND THEN lyzed then cell open. You then ran it on SDS PAGE + commassie staining but now you only found 2 bands. Based on the information, how many of the 3 are internal/integral proteins?

Could you however please explain the logic behind this a little bit. I'm getting a little confused. Thanks!

 

[sacjumpman]

Sorry, didnt see the other 2.

Questions regarding separation of proteins are actually quite straight forward, you just have to know the techniques and what is the basis of separation. 2 good ones to know will be differential centrifugation and density centrifugation. For example, if I give 2 proteins, one got MW of 900 and density of 2 g/cm3 and the other has MW of 650 and density of 2 g/cm3, which technique would you use?

Questions regarding identities of proteins are trickier, but its def. worth to learn it because it actually showws up on my DAT. For example, lets say you got a cell, lyzed it open and did SDS PAGE and commassie blue stained it and got 3 distinct bands. You then got another cell, from the same strain and then treat it with protease. After protease worked for a while, you added protease inhibitor AND THEN lyzed then cell open. You then ran it on SDS PAGE + commassie staining but now you only found 2 bands. Based on the information, how many of the 3 are internal/integral proteins?

So 2 of them are integral proteins?

These are the two that are unaffected by the protease?

And the third protein was peripheral which was cleaved by the protease? But then why wouldn't it show up on the plate at all afterwards?

I think I'm wrong actually, I'm trying to reason this out, but....

 

[jigabodo]

Could you however please explain the logic behind this a little bit. I'm getting a little confused. Thanks!

If you simply just lyze a cell and do SDS PAGE + staining to id proteins, you will be able to identify all the proteins of the cell... and in this case, there are 3 different proteins. (Well, usually with commasie blue staining you will get a crap load of different proteins showing up on the gel like a smere...because literally there are 10,000x different proteins in a given cell)

The purpose of doing a protease treatment THEN lyze the cell will ONLY take out the external proteins on the cell surface, because if the protein is integral/inside the cell the protease usually won't have access to chew them up because they are being protected by the cell membrane. So now, if you inhibit the protease, lyze the cell AND THEN run SDS PAGE + staining, only the proteins that are interior of the cell will show up, because they are protected by the cell membrane and therefore unaffected by the action of the protease. However, the external cell surface protein are not protected by cell membrane and therefore exposed, so it will be chewed up by the protease.

Hope it helps.

 

[sacjumpman]

If you simply just lyze a cell and do SDS PAGE + staining to id proteins, you will be able to identify all the proteins of the cell... and in this case, there are 3 different proteins. (Well, usually with commasie blue staining you will get a crap load of different proteins showing up on the gel like a smere...because literally there are 10,000x different proteins in a given cell)

The purpose of doing a protease treatment THEN lyze the cell will ONLY take out the external proteins on the cell surface, because if the protein is integral/inside the cell the protease usually won't have access to chew them up because they are being protected by the cell membrane. So now, if you inhibit the protease, lyze the cell AND THEN run SDS PAGE + staining, only the proteins that are interior of the cell will show up, because they are protected by the cell membrane and therefore unaffected by the action of the protease. However, the external cell surface protein are not protected by cell membrane and therefore exposed, so it will be chewed up by the protease.

Hope it helps.

You are the man. Thanks for all the help. It goes along way.

So just because you use a protease and it breaks down the peripheral proteins, then the proteins will NOT show up at all on the plate?

Are they just so small that they elute off quickly to the anode and don't really show up?

 

[jigabodo]

Sorry, just to add: sometimes the external protein will not be completly chewed up, but rather just partially chewed up. In such cases you will be able to observe a shift in one of the bands downwards, because the chewed up proteins are now lighter, and therefore travel faster.

 

[sacjumpman]

Sorry, just to add: sometimes the external protein will not be completly chewed up, but rather just partially chewed up. In such cases you will be able to observe a shift in one of the bands downwards, because the chewed up proteins are now lighter, and therefore travel faster.

Perfect.

That is what I had expected, and that's what threw me off a bit.

So for example, in the first one without the protease, it will be 3 bands.

Then use protease. The 1 peripheral protein may be broken into, say, 2 bands, and both will travel farther. And 2 of the bands will remain unchanged (these are the integral proteins).

 

[sacjumpman]

Sorry, just to add: sometimes the external protein will not be completly chewed up, but rather just partially chewed up. In such cases you will be able to observe a shift in one of the bands downwards, because the chewed up proteins are now lighter, and therefore travel faster.

I wonder if there is anything we should know about Western, Souther, Northern blot?

Besides very simply what they detect...

Also, what about microarrays? I guess I get the basic concept but time is tight right now, and I debate if I should learn more in depth?

 

[sacjumpman]

Sorry, just to add: sometimes the external protein will not be completly chewed up, but rather just partially chewed up. In such cases you will be able to observe a shift in one of the bands downwards, because the chewed up proteins are now lighter, and therefore travel faster.

By "downwards" you mean towards the anode, correct?

Away from the initial well?

Sorry, just to clarify. I always picture it moving upwards, but just want to make sure I got it straight.

 

[jigabodo]

By "downwards" you mean towards the anode, correct?

Away from the initial well?

Sorry, just to clarify. I always picture it moving upwards, but just want to make sure I got it straight.

Yes, more towards anode is probably a better explanation.

 

[jigabodo]

I wonder if there is anything we should know about Western, Souther, Northern blot?

Besides very simply what they detect...

Also, what about microarrays? I guess I get the basic concept but time is tight right now, and I debate if I should learn more in depth?

Don't bank on what I said, but microarray problems did not show up on my DAT. However, a similar but more complicated problem regarding protein identity like the one we just discussed showed up on my exam.

 

[sacjumpman]

Don't bank on what I said, but microarray problems did not show up on my DAT. However, a similar but more complicated problem regarding protein identity like the one we just discussed showed up on my exam.

I've read rumors about that one, along with the FRAP one. Not specifics, but just general stuff like we've discussed.

I just hope that I can reason it out because those that have taken it told me it was difficult to figure out the question even after they got home with google at their disposal.

 

[craptastic]

are u serious? problems more difficult than this? And we're supposed to reason this out in 1 minute? wow...

 

[jigabodo]

I've read rumors about that one, along with the FRAP one. Not specifics, but just general stuff like we've discussed.

I just hope that I can reason it out because those that have taken it told me it was difficult to figure out the question even after they got home with google at their disposal.

Don't stress. You are already a lot more prepared than me taking the DAT 2 weeks ago at those topics seeing how you can answer these questions really well. Have faith in yourself and I am certain that you will be able to score even higher than me on bio.

 

[sacjumpman]

Don't stress. You are already a lot more prepared than me taking the DAT 2 weeks ago at those topics already seeing how you can answer these questions really well. Have faith in yourself and I am certain that you will be able to score even higher than me on bio.

Hey, I appreciate it. Test is on Sat. So I've been working on just getting my confidence up. So a comment like that goes a long way. 😉

 

[jigabodo]

are u serious? problems more difficult than this? And we're supposed to reason this out in 1 minute? wow...

Yea...I think that problem alone took me around 5 minutes, and I still wasn't exactly sure if I got it right or not. I actually finished the TS section rather early so I had quite some time to solve these odd balls...

Fortunately, I only had 2 or 3 extremely difficult questions like this showed up on my DAT, although it definitely threw me off. My suggestion is that if you feel like you cannot finish it in 1 min, just guess, mark, move on and come back when you have time.

 

[sacjumpman]

Yea...I think that problem alone took me around 5 minutes, and I still wasn't exactly sure if I got it right or not. I actually finished the TS section rather early so I had quite some time to solve these odd balls...

Fortunately, I only had 2 or 3 extremely difficult questions like this showed up on my DAT, although it definitely threw me off. My suggestion is that if you feel like you cannot finish it in 1 min, just guess, mark, move on and come back when you have time.

See, normally this would always be my approach on any other exam. If it throws me off, skip it and come back.

However, on my Achiever exams, I always have only about 3 mins left at the end, so I've decided that it may not be good to just quickly mark it.

I'm wondering if Achiever just moves slower because of the heavy calculations in Gchem...

But yeah, I was trying to decide my strategy for these oddball reason-it-out style questions and still don't know how I'll tackle it.

Did you have extra time on your Achiever tests, jigabodo?

 

[poc91nc]

Just to add...it might be helpful to understand why the proteins seperate soley on the basis of mass in SDS-PAGE. The function of SDS (dentaturing, charge, etc)

Why we add a reducing agent (DTT/BME)?

PCR is definitely fair game as well. hmmm...taq polymerase/denaturing step all that jazz...

Monoclonal vs polyclonal antibodies.

Transfection techniques.

Recombinant DNA/proteins/reporter genes

Just shooting from the hip...so don't lynch me if anybody thinks this stuff isn't important.

If anybody is really paranoid about oddball Bio topics with respect to experimental biology (assuming you have time)....Chapter 8 of Molecular Biology of the Cell has everything you ever wanted to know about experimental biology but were afraid to ask. This kind of stuff just isn't covered in Schaums or any of the other popular guides...so, if you haven't had much experience with this stuff it might be a worthwhile time investement.

 

[jigabodo]

See, normally this would always be my approach on any other exam. If it throws me off, skip it and come back.

However, on my Achiever exams, I always have only about 3 mins left at the end, so I've decided that it may not be good to just quickly mark it.

I'm wondering if Achiever just moves slower because of the heavy calculations in Gchem...

But yeah, I was trying to decide my strategy for these oddball reason-it-out style questions and still don't know how I'll tackle it.

Did you have extra time on your Achiever tests, jigabodo?

On average, I have around 5 minutes left when I finish Achiever TS. I think I had more time for the 1st and 3rd test, but BARELY finished ontime for the second one.

On the actual DAT, if you really know your stuff on TS, you shouldn't be spending more than 30-45 seconds per question on GC and OC because they are very straightfoward and require minimal calculations. Sacjumpman, to be honest with you I think you will be able to do very well on GC/OC.

However, bio is a different story. There are around 20-30 questions that are the "you know it or you don't" type, which most people should have no problem with. The only difficult problems are the random ones and the ones that ask about techniques/applications.

Given the level of difficulty of GC/OC, I really think you will have more than enough time to go back to those weird questions once you are finished, don't stress too much about it.

 

[sacjumpman]

On average, I have around 5 minutes left when I finish Achiever TS. I think I had more time for the 1st and 3rd test, but BARELY finished ontime for the second one.

On the actual DAT, if you really know your stuff on TS, you shouldn't be spending more than 30-45 seconds per question on GC and OC because they are very straightfoward and require minimal calculations. Sacjumpman, to be honest with you I think you will be able to do very well on GC/OC.

However, bio is a different story. There are around 20-30 questions that are the "you know it or you don't" type, which most people should have no problem with. The only difficult problems are the random ones and the ones that ask about techniques/applications.

Given the level of difficulty of GC/OC, I really think you will have more than enough time to go back to those weird questions once you are finished, don't stress too much about it.

Sounds good. Then that will be my approach, I won't get too caught up with the trouble ones in bio. And will plan to come back. 🙂 Thanks for the help.