Dear all,I am a doctoral student of Xiamen University in China....

YL
Yingying Lin
Dec 23, 2015
protocol Protocol: Polysome Profiling Analysis
Dear all,
I am a doctoral student of Xiamen University in China. Recently, our lab bought a BioComp gradient machine. And I have done several times of polysome profile of MCF-7 cell line in order to set up the technique. However, the result didn't comes out good. Here I present a grap
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laura GA Answered Mar 22, 2016

isciii

Dear Ivan,
Thank you for the protocol. I have tried it with MCF7 cells (4 of 15-cm dishes). After of lysis, the OD 260 is around 2. What is the problem?. Also, I tried it with 293T cells (10 of 10-cm dishes) and the OD 260 was 1.5 . I believe the problem doesn´t the cell number because I have a lot of cells. When I run these RNA samples in a sucrose gradient I get a good profile but with low OD 254. Then, the problem is the celular lysis?
Best
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Yingying Lin Answered Dec 25, 2015

Xiamen University

Nevermind,you've helped me a lot! Many thanks!
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Ivan Topisirovic Author Answered Dec 25, 2015

Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

If you followed the protocol, then then the only thing I can think of is that this could be due to the way you make the gradient or how the collector is set up, so I'd suggest you contact Biocomp. I can't help you any further as I have no idea what's going on. Never seen a profile like this. Good luck with the experiments and sorry I couldn't help.
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Yingying Lin Answered Dec 25, 2015

Xiamen University

Dear Ivan,
Today, I did another polysome profile according to the protocol of jove paper, I used 10OD 260(500 uL) RNA sample to 5~50% sucrose gradient and centrifuge at 36,000 rpm for 2 hr, but the result still not good. Accidently, I cut the impurity peak, but the wave after 80S didn't go down, I can't figure out what's wrong with it, can you help me and give me some advice? Many thanks!


Best regards,

Ying
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Yingying Lin Answered Dec 23, 2015

Xiamen University

Dear Ivan,
Thank you very much! I am so embarrased to say that I saw the sentence and it mentioned 500 ul of the sample, for the engineer who set up our machine told me to add to full of the centrifuge tube, so for several times I only load 300 ul of the sample. So I see, maybe I should just remove 500 ul of the gradient and add 500 ul of the sample. Thank you again for your advice!


Best wishes,

Ying
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Ivan Topisirovic Author Answered Dec 23, 2015

Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

I have never seen a profile like yours so it's hard to say what you're doing wrong.

Yes I would load 10 OD from 260, and if you make a gradient using Biocomp gradient master you just have to make sure that it's continuous 5-50% (you can read the manual if you're confused re: zonal vs isopycnic centrifugation and it's crucial that you use rate zonal and not ispycnic caps). These are two things that I would try, and see whether they'd help.

If you followed the JOVE video and protocol it tells you exactly how much to load:

"Transfer ultracentrifuge tubes containing sucrose gradients in pre-chilled rotor buckets. Remove 500 µl from the top of sucrose gradients. Adjust lysates so that they contain the same OD (10-20 OD at 260 nm) in 500 µl of lysis buffer (described in step 2.5) and load them onto each sucrose gradient. Note: It is important to immediately load lysates on the gradients, as this will critically improve the quality of polysome preparations."

So it seems to me that you could follow it a bit more carefully, and according to my experience if you can't find this information in the protocol that's written as clearly as JOVE or the one here, then there may be a problem of how careful you are when you do the experiment. So try to be more careful.
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Yingying Lin Answered Dec 23, 2015

Xiamen University

Dear Ivan,
Thank you for your fast reply. Actually, I have seen the jove video you mentioned. And I done the polysome profile according to it as well, because I see your protocol is similar as that. You mean I overload the RNA to the gradient, I don't understand what is 10OD at 254 mean, I have measure the RNA with nanodrop, the OD 260 is 18.198, should I dilute the sample to adjust the OD260 to be 10, and how much volume of the sample should I load to the gradient? We use the sw41i rotor. On the other hand, there was something wrong with my gradient, what do you think is wrong?
Many thanks!

Best regards,

Ying
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Ivan Topisirovic Author Answered Dec 23, 2015

Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

Looks like you are overloading the gradient and/or that there be something wrong with your gradients. What we typically load is about 10OD at 254nm. Note that we use Biocomp gradient master but we fractionate using ISCO collector and not Biocomp (so we can't advise on Biocomp), but this shouldn't be an issue. This should help, as here you have the detailed protocol (with things that may go wrong) and whole video on how to do it.

http://www.ncbi.nlm.nih.gov/pubmed/24893926

Hope this helps, and if not I'll transfer you to Morita as I haven't done one of these in a while.

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