Science Buddies: "Ask an Expert"

Hi All,

I stumbled upon some more questions that I have about molecular biology protocols. I can't seem to find very good texts on these subjects, as the procedures are probably not used often or are too advanced for Wikipedia. Can anybody explain the following lit of methods to me and how they work? Thanks!

"Biochemical analyses of human telomerase by glycerol gradient sedimentation and gel filtration have shown that telomerase resides in a large complex of approximately 1-2 MDa.
I understand what the statement is going at, but I have no idea how glycerol gradient sedimentation and gel filtration work.

"We purified TERTcomplexes from human cells using a Dual Affinity Chromatography Strategy coupled with mass spectrometry."
Same deal here; I don't know what Dual Affinity Chromatography Strategy is, and while I understand the procedure for mass spectrometry, I'm not sure how it helps in purifying the TERT complexes (TERT is a reverse transcriptase molecule).

"To identify novel protein components of the telomerase complex, we purified TERT protein complexes using a modified TAP tag approach."
I don't know what TAP tags are...

Sorry for this mountain of questions!
Thanks,
Eric

Eric,

I am afraid I don't know the first thing about these procedures, and am leaving for a conference today. Perhaps one of the other experts can help--but if you are interested in these sorts of projects, I would really encourage you to find a mentor, because I'm fairly sure that you will need some advanced, expensive equipment to do the procedures you have listed. If you can find a mentor, they can probably explain these processes to you (or have a graduate student or a post-doc explain it).

I will ask the other experts to take a look at this post, but I'm not sure we have anyone in that field here over the summer.

Hi Mellisa,

Thank you for your advice! Yes, finding a specialist will probably yield more detailed responses.

Meanwhile, I have an additional more open-ended question still in the molecular biology category. I have recently read some journal articles discussing the identification of some protein required for some holoenzyme process.
I can see how this can be done- create knockout cells that do not exhibit the protein and observe adverse/benign/beneficiary results. Knowing this, how do researchers go about determining the modus operandi of a protein? For example, the structure, function, and procedure of the DNA polymerase enzyme is clearly well-defined.

How would one characterize the molecular pathway of a given enzyme or protein?

I hope this question isn't to out-of-scope.

Thanks,
Eric

Eric,

It's definitely not out of scope, but unfortunately we don't seem to have anyone around this summer who studies molecular biology--which means that I can answer you with vague recollections from my biochemistry/genetics courses, but that may or may not be very helpful.

The honest answer to your question is that it probably depends a lot on the protein. It is possible to stain embryos during development so that you can visualize where on an animal a particular developmental gene is being expressed. For example, if you stain a drosophila embryo with an antibody that targets 'eyeless', you will see that gene being expressed near the front of the head where the eyes should be. To some extent, you can tell which genes regulate which other genes by examining the knockout mutants carefully. For example, you could have a gene for eye location and a separate gene for eye pigment. If you prevent expression of the eye location gene but you still get pigment where the eyes would be, the first gene probably isn't in the regulatory pathway of the other. On the other hand, if you get no eye and no pigment, you can be fairly certain that the eye pigment gene needs the eye location gene to be expressed in order to function properly.

With enzymes, you can probably take a more biochemical approach and simply assay its abilities to bind with certain molecules or its ability to catalyze a reaction.

Nowadays, the functions of many proteins and enzymes in an organism can be guessed at because their functions are often conserved across many species--for example, the main gene that controls eye formation (and also part of head formation) is the same in cephalopods and humans!

Does that help any? I apologize if I'm not completely answering your question.

Hi Mellisa,

Thank you for your answer! so identifying a gene pathway boils down to observational research, either with knockout tests or flagging techniques.

Another question I have resides in protein purification. Most proteins are not expressed throughout the entire cell cycle, so how would one identify a protein in the cell processes? I imagine that some reagent would have to be added to cells to lock them in such a state so that there is enough time to identify the proteins through chromatography or mass spectrometry?

Thanks!
-Eric

Actually, if you know what gene goes with the protein of interest, you can just knock them into a bacterial (or some other) cell and genetically program the bacterial cell to make loads of protein for you. That's how we now get insulin to treat diabetics! (Interestingly, I just checked wikipedia to be sure I was right, and it turns out that we've now moved insulin production into safflower plants.)

Then you have lots of the desired protein you can do mass spectroscopy, etc. on.

Hi Eric,

I am a chromatography specialist, and I am checking in to answer your questions on protein purification using chromatography, and try to help with the molecular biology questions. Summer is an excellent time to do your background reading and narrow down your science project to something that is feasible with your available resources. You are asking about basic techniques that are used to purify biomolecules.

Glycerol sedimentation is a technique that can be used to separate very large molecules based on their density and requires an ultracentrifuge. This is a good technique to concentrate a sample.

Liquid chromatography is a basic technique which involves packing a chromatography resin in a column, and then applying a sample, which partitions into different fractions based on interaction with the solid support. The sample is eluted with a mobile phase.

Gel filtration is a chromatography technique that separates proteins by their size. A size exclusion resin is packed into a column, the resin is equilibrated in a neutral pH buffer, and the sample is applied to the column. Large molecules flow through the column first followed by smaller molecules. The chromatographer collects fractions as they elute from the bottom of the column. Gel filtration is a fairly easy technique to do because it involves just one mobile phase for equilibrating and eluting the sample, however, a sample is always diluted using this technique, so it’s usually necessary to use other techniques that concentrate the sample.

Affinity chromatography is another chromatography technique that involves using a resin that has a ligand covalently attached to the resin beads. Usually the ligand is an antibody that will bind specifically to the protein to be purified. The sample is applied in one buffer, and then contaminants are washed through using the same buffer, and the sample is then eluted using a buffer that will break the antigen-antibody bond. Since TERT binds to RNA, it might be possible to use an affinity support coupled to RNA to purify the enzyme.

The TAP-tag method involves cloning a protein with a tag that includes 4 different interaction sites that can be used for selective purification of the tagged protein away from other proteins in the sample. The details of this protocol are described in the paper below:

http://www.nature.com/nbt/journal/v21/n ... bt773.html

There are many other techniques that are used to purify protein complexes. Unless you have access to a research laboratory, one of the challenges in doing science fair projects is that you will be limited by your equipment. Sedimentation techniques do require ultracentrifuges, but chromatography techniques can be done using either chromatography systems or by simple gravity flow techniques that can be done with glass columns. What type of lab will you have access to for this project?

Characterizing the molecular pathway of a specific protein or enzyme would probably not be a suitable topic for a science fair project. Usually this type of work is done one study at a time by a team of scientists over a period of years. I think you should continue with your background reading and try to select a question or topic that you could answer with one or more experiments. Do you want to clone a purify a protein complex?

I don’t know what your background is, so do let me know if you need additional detailed information about any of these topics. What type of project are you interested in doing?

Donna Hardy

Hi Donna,

Thanks for the lengthy, detailed explanation! I have a couple more questions I would like to ask-

Affinity chromatography is another chromatography technique that involves using a resin that has a ligand covalently attached to the resin beads.

For the affinity method, is it similar to western blotting then, except an electrophoresis matrix is used instead of a resin?

and just for clarification, do all of the methods you mentioned before (excluding size exclusion resin and sedimentation) require some sort of protein-specific or RNA-specific antigen in order for hybridization to occur? If so, then how are these "probes" engineered?

My research background covers telomeres, telomerase, and a little bit of oncology. I would like to compare the effects of telomerase biogenesis inhibition through several protein/holoenzyme components (e.g. dyskerin, ponin, TERC) particularly in cancer cells for my experiment, so that was why I was interested in the characterization of proteins and their pathways in the cells (to identify inhibition candidates). What kind of equipment do you think I would require? I understand that it is a very complex project, so I am trying my best to acquire a research lab, but often labs don't have the time to support an independent research project. For a telomerase biogenesis inhibition project, I am guessing I would require the basic equipment necessary to sustain cell cultures, equipment for drug application (retrovirus, electroporation, etc.), and some assay to determine the health of subject cells.

In the event that I cannot get access to a lab, I will have to fall back on a simpler experiment- The supplies I have access to are
-high school lab glassware
-general chemistry tools
-medium-grade centrifuges
-basic equipment/reagents, as long as high-end equipment or multiple specialized reagents concerning telomerase are involved. Even then, I require help from an independent research institution if I plan on using vertebrate test subjects or human tissue (cancer cells), as regulated by science fair rules.

As you can see from the relative complexity of the experiment, it is one of my biggest concerns this year to gain access to a lab equipment, if not a mentor

Thanks,
-Eric Jang

Hi Eric,

You are very perceptive about the similarities of Western blotting and affinity chromatography. However, Western blotting is an analytical technique; a sample is run on an electrophoresis gel to separate the proteins, then it is transferred to a membrane and the specific protein of interest is detected using an antibody. So this is a good way to determine if the protein you are looking for is present.

With affinity chromatography, a ligand molecule is covalently attached to a beaded resin, which is packed into a chromatography column. The sample containing the molecule that will interact with the ligand is passed over the column, and the column is washed to remove other proteins. The specific protein is then eluted using a buffer that will disrupt the bond between the ligand and the sample protein. Common ligands are antibodies, which are used to capture specific proteins. The difference is that a quantity of protein is obtained that can be used for other experiments.

Other chromatography techniques such as size exclusion, ion exchange, hydroxyapatite and hydrophobic interaction are more general and will separate proteins based on different characteristics. Typically several steps are used in the purification of a specific protein from a complex mixture. It is helpful if someone has published a procedure for purifying a protein of interest. Developing a protocol to purify an unknown protein can take months, or even years. There was a project at the recent ISEF show where a student had spent a year trying to purify an unknown protein, with no success. The student had done an excellent job of presenting her project, but, of course the project did not win an award because there were no results. So, I would not recommend a project that would require purification of a novel protein.

For your project, you would need to be able to culture cells and have an assay specific for a specific enzyme pathway you are interested in. For this project it would be best to work in a laboratory doing similar research so they will have the cells and assay methods available. You also need a local mentor to help you with this project. You can contact local universities to ask for a referral or perhaps contact your local chapter of the cell biology society and inquire about availability of a mentor. A personal referral from a teacher might also help. You should also look for research grants that might be available to apply for so you can purchase lab supplies. If you can't find a mentor or a local research lab, then you will need to design your project to match your resources. Why don't you continue to look for research articles in your general area of interest and try to identify a specific pathway to work on and think of ideas for both types of research projects? When you find a research article, look through the materials and methods section and read the details of the equipment and protocols required for the experiment. That will give you an idea of what you will need. For the time being, concentrate on finding a local lab and mentor, but also consider the possibility of doing a less sophisticated project. For the science fair, you need to identify an experiment that you can do with whatever resources are available.


Donna Hardy

Hi Donna Hardy,

Thank you so much for the explanation! That may come in handy if I want to purify a large protein complex, but I will still need the matching anti-body, right?

I happen to have another off-topic question that I've been debating to myself over the past week:

As a background, I've spent quite some time searching up various faculty researchers from local labs that I wish to seek out for mentorship. I have read the sciencebuddies "How to Get a Mentor" section, and following that, I have sent emails to professors stating that I was interested in "doing research". However, all of the emails that I sent to them in this manner were ignored or promptly rejected. I tried a different approach, where I emailed the professor saying that I had some questions about their work, and if I could meet them in person.
Here are the pro's and con's of either approach, in my opinion:

Method 1: Tell the professor your accomplishments in their subject and add that you are interested in doing research in their lab.
Pro: I am able to attach a resume, state my scope of personal research in the field, and attempt to impress them beforehand.
Con: No reply or a response saying that I should look for a different lab. Often when I state my interests, they use it to their advantage to say that their lab doesn't do that kind of research.

Method 2: Tell the professor that you have a lot of technical questions you would like to ask them, and request to meet them in person to discuss their work. It turns out that I can actually find most of the answers myself, thanks to the internet, but the purpose of the email is to be able to meet them in person so I have a larger chance of acquiring a mentor.
Pro: Ambiguity of message will yield a larger chance of getting a reply (from my experience).
Con: The majority of replies are along the lines of "Sorry, I don't have time, but could you send me your questions over email?".

Of course, neither of these methods seem to work for me, as I have yet to receive a positive reply.

I am assuming that a marjority of the sciencebuddies experts are professors themselves, so it would be great if you could tell me what kind of email a professor, personally would rather get- an ambiguous one requesting an interview or a straightforward attempt to request a research opportunity? Both have their ups and downs, and as the number of labs that actually operate in the scope of my research are limited in this area, I don't want to jump the guns


Once again, thank you so much!
-Eric Jang

Hi Eric,

I worked with high school students in my lab as a PhD student and post-doc, and I have worked with high school and college students in my current research program.

It sounds like you are doing a good job of contacting possible mentors and introducing yourself. Keep it up! University faculty & researchers are typically very busy, and it is not unusual to get a delayed reply or no reply to an emal. So, don't be disappointed if you don't get immediate replies. An email response asking for you to send your questions by email is a decent success - follow up on that and see where it leads you.

A couple of additional tips - Read their recent papers and ask genuine questions that are specific to their work. If you can't get their papers, ask them for copies. If you ask them questions that you can answer using a web search just for the sake of getting their attention, then they may sense you are not being genuine, and they might not be as interested to discuss the topics with you and they might get the impression that you are not aware of the exact research questions being addressed in their laboratory.
Don't spent too much time telling them about your accomplishments. It's good that they know you have won certain awards or gotten good grades, but more important to them will be that you are reliable, hard working, enthusiastic, and eager to learn in their laboratory. One way to convince them of those qualities is to show in advance that you are willing to take the time to read and understand one of their papers, and to discuss it further with them.

That's my 2 cents. Good luck!

Chris

Just quickly to add to what Chris has already said--if you don't get a reply within two weeks, send another e-mail saying that you think your e-mail was overlooked. I know a lot of professors who glance at their e-mail, intend to reply to it, and then get distracted by something else and forget about it. I have collaborators that I regularly have to e-mail 2-3 times before I get a reply even on things that are really important to them. So, be persistent.

I would also try to aim for people whose research interests overlap your own. It's okay to say, 'This is what I'm interested in, but my interests are flexible and I think I could get interested in what you're doing in your lab'. Most professors don't have permits/resources to spend on things that are not in some way related to their research, so you may have to be a bit flexible about what you'd like to do unless you happen to have someone who is in your area who works on exactly what you want to work on.

Another way to go is to try to get into a program that sets high school kids up with mentors--I know they're out there, though they may be very competitive.

Hope this helps!

Hi Chris, Melissa,

Thanks for your advice! I will make sure to ask legitimate questions to prospective mentors.

As for the high school programs, I have looked into a lot of them in my local area. Most advanced research programs, however, are only applicable to rising juniors and higher, so I will have to wait until next year. Regarding the lab publications, I have accumulated a small "library" of journals from my mentors, which provide a good knowledge resource. I was reading through one of them today when I suddenly realized that I never bothered asking a question about something I saw in almost every lab report.

In telomerase-related publications, the researcher often performs a telomerase-level assay in what appears to be a southern blot. They present what appears to be gel assays and protein separation, but I am having difficulty interpreting the diagrams. I have tried asking these kinds of questions to the lab members themselves, but the only people that bother replying are quite terse with their responses and redirect me to other parts of the paper, so asking them takes awhile to extract information. The experts here on Sciencebuddies are much more in-depth with their responses Would somebody mind explaining to me what the data means on page 645-646? http://www.sciencemag.org/cgi/reprint/3 ... siteid=sci.

for diagram
A: it looks like they used a "PAGE" procedure to separate dual-affinity purified dyskerin? I'm not sure.

As for the rest of the diagram, I'm sure they prove the point that TCAB1 is indeed necessary for telomerase function, but kind of iffy on how those blots do so.
B:
C:
D:

pg. 646:
A:
B:

Sorry for this big clarification question- I'll try my best to provide what I know if I need to answer basic questions about telomerase for your reference.

Once again,
Thanks,
Eric Jang

I'm afraid this is over my head--perhaps Donna Hardy can help. But, have you looked at the supplemental information for the study? It includes details on the materials and methods, which may be the details you're interested in. You can find it here: http://www.sciencemag.org/cgi/data/323/5914/644/DC1/1.

Take a look at that and see if it answers some of your questions. If not, and if Donna doesn't show up in a few days, I'll take a closer look at it and see if I can help...but I can't make any promises.

Hi Melissa,

Thanks! I didn't know that there was a supplemental materials section- that will definitely help!
-Eric