[Demelerlab] Isolated lysosomes for AUC

Sun Jul 9 13:52:53 MDT 2023

Everyone:
The samples I picked up from Jake on Friday in Missoula made it safe &
sound to our -80 freezer with plenty of dry ice to spare, so they arrived
in good shape. I would like to schedule a zoom meeting between all of you
and my lab to discuss the experimental designs to be used. Andrij, Jake and
Matt: please send me your availability for this coming week.  We are
relatively flexible. I would like everyone in my lab to have a chance to
learn about your project, and contribute thoughts for a successful
experiment.

Thanks, -Borries

On Fri, Jul 7, 2023 at 4:23 PM Albright, Jacob <
jacob.albright at umconnect.umt.edu> wrote:

> Borries,
> I have registered and submitted a project request form on your website
> including all the details about our samples. Below you will find an OD
> image of a lysosome sample that was made along side the ones we’re sending
> with you. The sample used in the image was diluted by a factor of 4 because
> we needed a larger volume to run the OD. Also, I’m sending extra buffer
> (both H2O and D2O) incase the samples need to be diluted to reach that
> which was used to get the OD.
>
> Thanks,
> Jake
>
> On Jul 6, 2023, at 8:41 PM, Borries Demeler <demeler at gmail.com> wrote:
>
>
> Sounds good, Jake. We will also need you to register on the LIMS server
> and enter a project request with the details, so we can put you into the
> queue. You can register from this link:
> https://uslims.uleth.ca/uslims3_CCH/newaccount.php
> and then you can submit a project request form from the "Projects" button
> in the left bar (after logging into your new LIMS account).
>
> -Borries
>
>
> On Thu, Jul 6, 2023 at 7:50 PM Albright, Jacob <
> jacob.albright at umconnect.umt.edu> wrote:
>
> Yes, I can meet you outside Andrij’s office at 5. The samples will be in a
> well-insulated box with plenty of dry ice. I will have all the details of
> the samples for you tomorrow as well.
>
> Thanks again,
> Jake
>
> Get Outlook for iOS <https://aka.ms/o0ukef>
> ------------------------------
> *From:* Borries Demeler <demeler at gmail.com>
> *Sent:* Thursday, July 6, 2023 5:31:06 PM
> *To:* Albright, Jacob <jacob.albright at umconnect.umt.edu>
> *Cc:* Sydor, Matthew <matthew.sydor at umconnect.umt.edu>; Holian, Andrij <
> andrij.holian at mso.umt.edu>; Amy Henrickson <amy.henrickson at uleth.ca>
> *Subject:* Re: Isolated lysosomes for AUC
>
> OK, pack a lot of dry ice, and I will pick them up tomorrow around 5
> pm, will stop at Andrij's office, is that ok?
> -Borries
>
> On Thu, Jul 6, 2023 at 3:35 PM Albright, Jacob
> <jacob.albright at umconnect.umt.edu> wrote:
> >
> > Borries,
> > It would nice if we can keep them frozen until use since that is how we
> have been handling them so far. Can we send them with you on dry ice?
> >
> > Either way they will be ready at your earliest convince, tomorrow, as
> they will be done today.
> >
> > Jake
> >
> > On Jul 6, 2023, at 3:23 PM, Borries Demeler <demeler at gmail.com> wrote:
> >
> > When do you expect to be ready with the sample, and how do they need
> > to be stored? Is 4C acceptable? If so, I can pick them up anytime and
> > store them in my fridge until I leave.
> >
> > -Borries
> >
> > On Thu, Jul 6, 2023 at 3:21 PM Albright, Jacob
> > <jacob.albright at umconnect.umt.edu> wrote:
> >
> >
> > Borries,
> > It’s funny you should ask. The samples are being prepared currently. Do
> you have a rough estimate as to when you will be by?
> >
> > Jake
> >
> > On Jul 6, 2023, at 3:18 PM, Borries Demeler <demeler at gmail.com> wrote:
> >
> > Andrij, Matt and Jake:
> > Any updates on whether you will have samples for me to pick up
> > tomorrow? I am planning to leave early Saturday to drive back to
> > Lethbridge and could take some along. If it doesn't work out this
> > time, I can take some along the next time I am in town, which will be
> > July 20-23. Just let me know.
> >
> > Thanks! -borries
> >
> > On Tue, Jun 27, 2023 at 4:45 PM Borries Demeler <demeler at gmail.com>
> wrote:
> >
> >
> > Ahh, thanks for reminding me about the volume! Each sample should be 0.5
> ml in volume. In terms of concentration, we need about 0.5-0.9 OD at
> whatever wavelength between 220-300 you want to use, but make sure your
> buffer has no or little background absorbance at that wavelength.
> >
> > -Borries
> >
> > On Tue, Jun 27, 2023 at 4:32 PM Albright, Jacob <
> jacob.albright at umconnect.umt.edu> wrote:
> >
> >
> > Borries,
> > That all sounds doable. Yes, each sample will have the same lysosome
> concentration. What should the volume/sample be?
> >
> > We will get back to you about the ice.
> >
> > Jake
> >
> > On Jun 27, 2023, at 3:57 PM, Borries Demeler <demeler at gmail.com> wrote:
> >
> > Excellent timing, Jake, my schedule has changed by a few days, so you
> have until July 7th or 8th. I'll be in Missoula next week, and can take
> your samples with me. Right now, it looks like I will be leaving Missoula
> again on the 8th. I will contact you guys next week with the exact plan.
> Yes, a 90%, 50% and 0% D2O buffer would be great. Also, it would be good if
> I knew the OD at multiple wavelengths. If you could get a good
> spectrophotometer reading (staying below 1 OD between 230-300 nm) of your
> sample that would be optimal. This would be the concentration after
> dilution into the target buffer, so dilute each one the same, OK? Can we
> leave the sample on wet ice, or do you expect it needs to be on dry ice? I
> can schedule an experiment for Monday, July 10th, so the sample would need
> to be stable until then. Please create an account on our LIMS system and
> submit a project request (
> https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fuslims.uleth.ca%2Fuslims3_CCH%2Fnewaccount.php&data=05%7C01%7Cjacob.albright%40umconnect.umt.edu%7C7db841c3c1ca405e04af08db7e791a28%7C68407ce503da49ffaf0a724be0d37c9d%7C0%7C0%7C638242830838024280%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=wWhvqtqjSxeFz7LYFhwyAQ9thsdY%2BxQ%2F%2FLRkHhn5baQ%3D&reserved=0
> <https://uslims.uleth.ca/uslims3_CCH/newaccount.php>) so we get it into
> our system. Matt should remember how to do that.
> >
> > Regards, -Borries
> >
> >
> >
> >
> >
> >
> > On Tue, Jun 27, 2023 at 3:37 PM Albright, Jacob <
> jacob.albright at umconnect.umt.edu> wrote:
> >
> >
> > Hello Borries,
> > We are planning to prepare a lysosome sample this week for you to run
> AUC with. I believe you said you could pick it up on July 4th. Is that
> still the plan?
> >
> > We ordered some D2O and would like to make sure the sample is prepared
> correctly. I am isolating a brand new sample for this and will make 2
> versions of our cytosolic buffer. The only difference being one is made
> with H2O and one with D2O. Considering what we talked about, I am thinking
> of suspending the sample in a 50% mixture of both buffers and keeping it
> fairly concentrated (100 uL of each buffer) and then including additional
> buffer for you to dilute with as needed. We could send up to 9 mL of each
> buffer. Would that work? I’m not sure how much volume you will need for the
> AUC.
> >
> > I will also include the sample weight. It will probably be a few mg.
> >
> > Jake
> >
> >
>
>
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://www.biophysics.uleth.ca/pipermail/demelerlab/attachments/20230709/60e0c768/attachment-0001.html>