[Demelerlab] Isolated lysosomes for AUC

Sun Jul 9 14:42:01 MDT 2023

Borries,
I have meetings with Jake and Matt on Thursdays from 10-12 so somewhere then would be convenient.
Otherwise Wednesday morning is available at my end.
Andrij

From: Borries Demeler <demeler at gmail.com>
Date: Sunday, July 9, 2023 at 1:53 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>, "demelerlab at biophysics.uleth.ca" <demelerlab at biophysics.uleth.ca>
Subject: Re: Isolated lysosomes for AUC

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<mailto: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<mailto: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<mailto: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

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________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Thursday, July 6, 2023 5:31:06 PM
To: Albright, Jacob <jacob.albright at umconnect.umt.edu<mailto:jacob.albright at umconnect.umt.edu>>
Cc: Sydor, Matthew <matthew.sydor at umconnect.umt.edu<mailto:matthew.sydor at umconnect.umt.edu>>; Holian, Andrij <andrij.holian at mso.umt.edu<mailto:andrij.holian at mso.umt.edu>>; Amy Henrickson <amy.henrickson at uleth.ca<mailto: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<mailto: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<mailto: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<mailto: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<mailto: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<mailto: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<mailto: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<mailto: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<mailto: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
>
>

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