Experimenting on Actin & Myosin: Proteins, Tests & Results

[MusicMonkey]

What are some possible practical experiments that would enable the understanding of composition, structure and/or functions of the protein preparations that were made (actin and myosin). How would these experiments apply to studying actin and myosin.

I know that SDS-PAGE and Bradford Protein Assay could be used and I understand how they would apply to studying actin and myosin but what other procedures would also apply.

I am not sure if these two could apply:
1. Proton NMR, Mass Spectroscopy, IR Spectroscopy
2. MRI

Please help. Thank you in advance.

 

[noobie]

Not sure any of those techniques can be successfully applied to study actin or myosin.
I'm sure there are research groups trying though. My guess is that some type of microscopy technique is your best bet for these fibrous proteins (AFM, cryo EM, etc).

 

[MusicMonkey]

Thank you noobie for the suggestions of AFM and cryo EM. I will look into those two techniques.

Does anyone know if the 2 suggestions that I made could be applied to study actin or myosin?

Thank you.

 

[noobie]

MM, I'm not sure what type of info you're trying to get on actin/myosin. Simple proton NMR is limited for smaller molecules. There are of course researchers using multidimensional NMR for larger biomolecules but I'd imagine that it'd be hard to do for fibrous protein like actin unless you were only looking at the monomeric form. In any case, the proton NMR spectrum would be too complicated to resolve unless you did some isotope labelling. I'm doubting you could learn anything interesting about actin/myosin using NMR at least simple NMR expts.

Mass spectrometry & IR Spec - again these methods are not going to be able to resolve a system as complicated as actin/myosin.

MRI is really the same thing as NMR except it's mainly used on patients. It's an imaging technique and I don't think the resolution is nearly good enough to discern anything at a molecular level.

Perhaps, if you clearly state what type of info you're trying to get it'd be easier. I've used all those techniques before except for MRI.

 

[kalladin]

How's about Xray crystallography?

 

[noobie]

As far as I know, fibrous proteins are tough to crystallize. For x-ray studies you need a 3-D crystal. Cryo-EM is ideal because you only need a single layer of the stuff. Plus when you do x-ray, you always risk that the structure you're solving is not biologically relevant. You might be able to crystallize actin/myosin but will it give you any insight to what's going on inside the cell? Another very simple yet useful technique is a fluorescence microscope. I would use that before any other technique.

 

[MusicMonkey]

Basically this assignment calls for further investigation of the differences in the two characteristics of the muscle protein preparations. We need to describe what kind of information it gives, how it works and indicate its application to muscle (how it would be applied to studying actin and myosin). Thank you very much for your continued help.

 

[MusicMonkey]

The two techniques that were already used while doing this experiment were the Bradford Protein assay and the SDS-PAGE.

 

[MusicMonkey]

Also are SPR and Cryo-EM the same thing? Does Cryo-EM give the same information as AFM? Thank you

 

[MusicMonkey]

I have just been reading more about Cryo-EM and that is definitely one of the techniques that I will be using but I still need one more. Would Cryo-EM and AFM give the same information? Are they the same techniques?

 

[noobie]

AFM and cyro EM are not the same techniques but they are both imaging techniques. I actually don't know which one will give you a better resolution (w/ AFM it depends a lot on the tip that you're using). Most of the techniques that have been mentioned are fairly sophisticated and maybe overkill for what you need. Bradford and SDS are fairly simple analytical tools. You might want to look at some blotting techniques using antibodies (Northern, Southern? can't remember which). I personally think a fluorescence microscope would be good. You can label the myosin and track its movement.

 

[MusicMonkey]

The assignment is also being marked on originality of the answer and the course is covering fluorescence microscopy so something like Cryo-EM is a technique that would be an original type answer. If AFM and Cryo-EM are not the same techniques and they do not work in the same way then I think I will include those two in the report. If there is any other technique that you can think of please suggest them otherwise I will use AFM and Cryo-EM. Thank you once again for your help.

 

[kalladin]

Don't we already know what actin and myosin does? and how it confers to the sarcomere and muscle contraction?

 

[noobie]

I would tend to say that there is no biomolecule that has been characterized well enough that we don't have anything more to learn about it. I mean the basic structure of DNA was solved some half a century ago and people are still studying the structure today.

 

[kalladin]

I guess... I'm just a minimalist :P This is bad, I must change my lifestyle!