[Demelerlab] AUC question

[Borries Demeler]

Hi Karen, Ajit and Margaret:

Sophia and Reece, with help from Amy, analyzed all of your samples and
Sophia prepared the attached analysis report for all of your PfDBP1
and CECR2 binding experiments. Please let us know if you have any
questions, we can also set up a zoom call to discuss these results, if
you would like to have a meeting, please send us your availability for
next week.

Thanks! -Borries

On Thu, Jun 22, 2023 at 2:32 PM Borries Demeler <demeler at gmail.com> wrote:
>
> Hi Karen,
> AUC cells are distinguished by their pathlength. 110 ul is fine if you quadruple the concentration to get the same OD. At low concentration you are much better off measuring in a 1.2 cm centerpiece (~ 500 ul) to make sure we see enough signal. You can have low concentration and you can have short pathlength, but you cannot have both, of course. Sorry if this wasn't clear.
>
> -Borries
>
> On Thu, Jun 22, 2023 at 1:21 PM Glass, Karen C <karen.glass at med.uvm.edu> wrote:
>>
>> Hi All,
>>
>>
>>
>> Sorry to only reply now, I was stuck in NIH study section meetings for the last two days. We sent 220 uL to conserve protein after Borries mentioned the 110uL cuvette (but gave some extra for you to check OD with). However, we weren’t aware of the min protein concentration needed for that volume. Can you please clarify what concentration we should have to use the small cuvette?
>>
>>
>>
>> The protein:ligand ratios were designed to have the ligand concentration above and below the Kd. Diluting the samples may disrupt the ratios so that the concentration is now below Kd. We will need to do some calculations to figure this out. Can you let us know how much buffer you added to each sample?  This may be just fine and we will wait to see how the experimental data looks.
>>
>>
>>
>> Thanks,
>>
>> Karen
>>
>>
>>
>>
>>
>> Karen C. Glass, Ph.D.
>>
>> Associate Professor
>>
>>
>>
>> Department of Pharmacology
>>
>> University of Vermont College of Medicine
>>
>> Firestone Medical Research Building, Room 362
>>
>> 149 Beaumont Ave
>>
>> Burlington, VT 05405
>>
>>
>>
>> Email: karen.glass at med.uvm.edu
>>
>> Phone: 802-656-5760
>>
>> Twitter: @KarenGlassLab
>>
>>
>>
>>
>>
>> From: "Singh, Ajit K" <ajit.singh at med.uvm.edu>
>> Date: Tuesday, June 20, 2023 at 6:19 PM
>> To: "Henrickson, Amy" <amy.henrickson at uleth.ca>, "Glass, Karen C" <karen.glass at med.uvm.edu>
>> Subject: Re: AUC question
>>
>>
>>
>> Hi Amy,
>>
>> Thank you so much for the information.
>>
>> Happy to hear that OD worked for all the sample.
>>
>>
>>
>> Thanks
>>
>> Ajit
>>
>>
>>
>> Sent from Samsung Galaxy smartphone.
>> Get Outlook for Android
>>
>> ________________________________
>>
>> From: Henrickson, Amy <amy.henrickson at uleth.ca>
>> Sent: Tuesday, June 20, 2023 6:01:40 PM
>> To: Singh, Ajit K <ajit.singh at med.uvm.edu>; Glass, Karen C <karen.glass at med.uvm.edu>
>> Subject: Re: AUC question
>>
>>
>>
>> [ This message was sent from an address outside the Larner College of Medicine. Please exercise caution when clicking links or opening attachments from this source. ]
>>
>> Hi,
>>
>>
>>
>> Sorry about the confusion. I am not sure where the 220uL would have come from.
>>
>>
>>
>> We have prepared all of the samples for the project that involved the PBfDP1 protein, and the ODs still worked for all of them, so we have started the run. Tomorrow I believe we will do the CECR2 BRD project samples.
>>
>>
>>
>> Cheers,
>>
>>
>>
>> Amy Henrickson
>>
>> ________________________________
>>
>> From: Singh, Ajit K <ajit.singh at med.uvm.edu>
>> Sent: Tuesday, June 20, 2023 9:40 AM
>> To: Henrickson, Amy <amy.henrickson at uleth.ca>; Glass, Karen C <karen.glass at med.uvm.edu>
>> Subject: Re: AUC question
>>
>>
>>
>> Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>>
>>
>>
>> Hi Amy,
>>
>> I have been asking for the required volume of the sample for AUC and i have been given information to make 220uL volume.
>>
>> I hope while dilution to 470uL or 500uL should not make the OD below 0.2. I hope experiment to go well.
>>
>> I have also sent some sample for SAXS experiment trushar lab. I will let higor know to collect it.
>>
>>
>>
>> Thanks
>>
>> Ajit
>>
>>
>>
>> Sent from Samsung Galaxy smartphone.
>> Get Outlook for Android
>>
>> ________________________________
>>
>> From: Henrickson, Amy <amy.henrickson at uleth.ca>
>> Sent: Tuesday, June 20, 2023 11:34:01 AM
>> To: Singh, Ajit K <ajit.singh at med.uvm.edu>; Glass, Karen C <karen.glass at med.uvm.edu>
>> Subject: Re: AUC question
>>
>>
>>
>> [ This message was sent from an address outside the Larner College of Medicine. Please exercise caution when clicking links or opening attachments from this source. ]
>>
>> Hello,
>>
>>
>>
>> We require about 470 uL for AUC. There are times that we can change the pathlength and only need 110uL. However, in those cases, the sample needs to be highly concentrated because we are reducing the pathlength by 3, and therefore the signal we can detect is also decreased by a factor of three.
>>
>>
>>
>> Thank you for clarifying the volume. I have yet to thaw them, so I was just guessing. I will spec the samples and dilute as little as possible. I believe we should still be able to get some good results.
>>
>>
>>
>> Cheers,
>>
>>
>>
>> Amy Henrickson
>>
>> ________________________________
>>
>> From: Singh, Ajit K <ajit.singh at med.uvm.edu>
>> Sent: Tuesday, June 20, 2023 9:26 AM
>> To: Henrickson, Amy <amy.henrickson at uleth.ca>; Glass, Karen C <karen.glass at med.uvm.edu>
>> Subject: Re: AUC question
>>
>>
>>
>> Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.
>>
>>
>>
>> Hi Amy,
>>
>> Thank you for your email.
>>
>> I just wanted to know how much volume of the sample required for AUC because I have sent only 220uL of the sample.
>>
>> Please check the OD of the sample at desired wavelength mentioned in the protocol sent for the respective experiment.
>>
>> And then dilute it based on the requirement for AUC.
>>
>>
>>
>> Thanks
>>
>> Ajit
>>
>>
>>
>> Sent from Samsung Galaxy smartphone.
>> Get Outlook for Android
>>
>> ________________________________
>>
>> From: Henrickson, Amy <amy.henrickson at uleth.ca>
>> Sent: Tuesday, June 20, 2023 10:35:40 AM
>> To: Glass, Karen C <karen.glass at med.uvm.edu>; Singh, Ajit K <ajit.singh at med.uvm.edu>
>> Subject: AUC question
>>
>>
>>
>> [ This message was sent from an address outside the Larner College of Medicine. Please exercise caution when clicking links or opening attachments from this source. ]
>>
>> Hello Karen and Ajit,
>>
>>
>>
>> We are going to start some of the AUC experiments this afternoon. However, I noticed that the tubes only contain about 120uL of sample. I was wondering if you intended for us to dilute the samples to 500 uL.
>>
>> I noticed that one of the projects said that the OD was around 0.6 OD at 280nm; I wanted to double-check if that was at the 120uL or 500uL.
>>
>>
>>
>> I will spec everything before starting the AUC to ensure we are measuring at the correct wavelength. I wanted to ask you first.
>>
>>
>>
>> Thank you,
>>
>>
>>
>> Amy Henrickson
>>
>>
>>
>>
>>
>> ---
>>
>> Amy Henrickson, B.Sc
>>
>> Ph.D. Candidate
>>
>> Laboratory Manager for Canadian Center for Hydrodynamics
>>
>> Northwest Biophysics Consortium
>>
>> Alberta RNA Research and Training Institute (ARRTI)
>> Department of Chemistry and Biochemistry
>> University of Lethbridge
>> 4401 University Drive
>> Lethbridge, Alberta, Canada T1K 3M4
>>
>> Email: amy.henrickson at uleth.ca
>>
>> Phone: 403-329-2095
>>
>>
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