Hello everyone,
I recently started several polysome profiling experiment and something bugs me out.
Every protocols I found use 12mL tube.
They load their lysate containing ribosomes attached to mRNA and then ultracentrifugate for 2h at 260000 g.
The more ribosomes attached to a given mRNA, the more it migrates. By analysing fraction of the gradient you can determine if your mRNA of interest is efficintly translated or not.
The tube contain a 10-50% sucrose gradient. This way, heavy complexes don't migrate to soon (and don'taccumulate at the bottom of the gradient) compared to light complexes.
I only have 4mL tubes instead of 12mL. So the lenght of migration is divided by 3. Hence, I also reduced time of centrifugation by 3.
But, is this "rule of thumb" accurate ?
You should adapt the centrifugation time according to the clearance factor (k factor) and the maximal g force of the two rotors , which are given for each rotor by the manufacturer, and which you should be able to find on the Internet. Thus:
t4ml = t12ml x (k4ml/k12ml) x (gmax12ml/gmax4ml) for the same g force, i.e. 260 000 g in your case. See https://en.wikipedia.org/wiki/Clearing_factor for complete explanations.
But the reason why people use 12-ml tubes is because they afford a better resolution
Agree with Pierre Béguin that scaling down will lead to loss in resolution of the polyribosome fractions.
So, given Pierre Béguin's answer my centrifugation time should be in the range of 1h to 1h and 10 min.
t4ml = t12ml x (k4ml/k12ml) x (gmax12ml/gmax4ml)
t4ml = 90 min x (235 / 295) x (260000/260000)
K factor of 4mL Th660 rotor being 235 for a 5-20% sucrose gradient
K factor of 12ml SW41 rotor being 295 for a 5-20% sucrose gradient
and 90 min being the centrifugation time found in this protocol
Pringle, E. S., McCormick, C., & Cheng, Z. (2018). Polysome Profiling Analysis of mRNA and Associated Proteins Engaged in Translation. Current Protocols in Molecular Biology, e79.
I also found this 1h10min value with the formula t = K / S20,w
t = 235 / 200
where K is again the crearing factor of th660 for sucrose gradient and 1.7g/cm3 particles and S20,w the sedimentation coefficient of polysomes
This doc is really helpfull regarding centrifugation methods and theory.
https://thermofisher.co.nz/Uploads/file/Scientific/Applications/Equipment-Furniture/Practical-Techniques-for-Centrifugal-Separations.pdf
My last question will be why some protocols use fewer g force (https://bio-protocol.org/cn/e2126?action=Questions) or longer centrifugation time
(Article Cell-type specific polysome profiling from mammalian tissues
) with the same SW41 rotor ? Given the A254nm profiles looks alike. I suppose it's highly dependent on the tissu or cell line you're using right ?Thanks again for your help !
Sibnarayan Datta thanks for your answer.
To be clear, when you and Pierre Béguin talk about resolution, you refer to the A260 graph generated by a fractionator showing 40, 60, 80S and polysomes spikes ?
If so, I unfortunatly don't have one at my disposal... So I manually collet small fraction by hand from top to bottom. A long and painfull exercice hahaha !
I attached a file showing my "DIY A260 curve" generated by collecting 40 100µL fraction for a 4mL tube. Then I pooled every 5 fraction into 1 for downstream RNA extraction. You can see the RNA gel after extraction.
Dear Dr Phillipe,
by resolution I mean to say that with smaller volumes, all the RNA fractions are difficult to separate effectively in the sucrose gradient. Since you are using a manual fraction collector, it will be more difficult to uniformly collect fractions.
In fact, I did polysome experiments almost 10 years ago during postdoc and I remember that even following a published methodology, I had to optimize a lot, which it took months.
I am attaching the paper where we published our polysome analysis related work for your reference.
regards,
SD
Thank you for sharing your work Sibnarayan Datta
I realise now after several experiments that fraction collection will never be perfect when done by hand. It at least allows me to see monosome peak and to define where my polysome are broadly speaking.
I work on transitory trasfection to see the translation of just one protein of interest for which antibodies are messy. Hopefully, I have numerous controls other than A260 specter like deltaATG construct or other construct known to be translated.
In anyway, thanks again for the help you provided !
Hello Philippe Paget-Bailly , did you find a solution for your task? I have a similar problem, I need to get a polysome faction, but we don't have a gradient fractionator and 12 ml tubes, only available 5 ml tubes or 30 ml.
If you already found a solution for your task, could you share with me your experience?
Thank you,
Asya
Hi Asya Davidian ,
For a 4ml gradient centrifuge at 260000g for 1h15min.
The polysome profile was obtained by collecting 50 µL fractions.
I have pretty good results even by hand (graph attached).
So you end up with 80 fractions to measure at 260nm.
@ Philippe Paget-Bailly , Thank you! Polysome profile what you got looks great!
I did as you suggested 100 ul fractionation manually.
Do I need to measure OD260 directly from the fraction or should I first extract RNA with Tryzol and then measure it? Did you use Nanodrop for measurement?
I am just afraid that I don't have enough RNA concentration per fraction. What amount of RNA or protein did you use for loading on a sucrose gradient?
Thank you!
Hi Asya Davidian ,
you must measure OD before extraction. Yes I did it with nanodrop.
I realise I didn't gave you all infos sorry.
So for a 4 mL gradient:
- collect your 80 fractions of 50 µL
- measure OD 260 with nanodrop
- realise your OD260 graph on excel to see the different peaks of your polysome profile
- pool your fractions by ten, so you end up with only 8 fractions of 500µL
- Extract RNA
- RT-qPCR
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