[Demelerlab] AUC (sedimentation equilibrium experiments)

Thu Dec 28 09:38:11 MST 2023

Dear Dr. Demeler,
Thank you for the detailed information. I will try to ship it by today afternoon. I will ship the samples, today only, but I will share the aggregation plot by today evening or if morning. Please let me know if that is okay. I will make an account and upload all the data as soon as possible. Please let me know if that works for you.

Thank you for your time and consideration.
Jyoti

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________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Thursday, December 28, 2023 10:22:53 AM
To: Baranwal, Jyoti <baranwal at uthscsa.edu>
Cc: Gupta, Yogesh K <GuptaY at uthscsa.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: AUC (sedimentation equilibrium experiments)

*EXTERNAL EMAIL*

Hi Jyoti,
Thanks for the details. They should be uploaded into your LIMS account. I noticed you still have not created an account and uploaded a project request. Please do that at your earliest convenience.  Get started here: https://uslims.uleth.ca/uslims3_CCH/newaccount.php and read my earlier e-mail from 12/12 for further instructions.

Please keep in mind that whatever proteins we will measure, their contribution to the OD should be at least 0.3  OD, while the total OD for buffer and protein against water cannot be more than 0.9 OD at whatever wavelength you want us to measure. Also keep in mind that molar concentration should be the same in each condition so the measurements can be compared. Choose the wavelength to be used for measurement based on these criteria.

Yes, we will need about 15 ml of each buffer in addition to the protein samples. It may be best if you just send the protein in concentrated form and we will make the dilutions in our lab.

Please send detailed instructions and if possible send the samples today to 1907 Hollis Street, Missoula Montana, 59801,  with overnight Fedex.
We are leaving Tuesday morning back to Lethbridge and shipping to Canada is always very complicated, so it is better if you send everything to my address in Montana before we leave to go back to Canada on Tuesday early morning. Make sure the samples arrive no later than Monday so we can still take them back with us  to Canada - I will not be in Montana until the end of February and your samples would go to waste if they arrive late.
Let me know if you have any questions, and send me the tracking number.
Thanks! -Borries

On Wed, Dec 20, 2023 at 11:30 AM Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>> wrote:
Dear Dr. Demeler,
I did the buffer absorption profile and have shared it in this email as a PowerPoint. I have checked the buffers listed in the previous email and also the low Tris (15 mM) and Low Sodium Acetate buffer (15 & 25 mM) buffer as suggested by you. I have checked the UltraScan Buffer absorption profile to decide on Tris and Sodium Concentration to 15, 15, and 25 mM, respectively. My buffer absorption profile suggests the same. In this, I can see that Bis-Tris has high absorbance values. Based on this data, I am planning to use only MES buffer not the Bis-Tris for both the pH 5.6 and 6.5.
Based on the profile Buffers I am planning to use for my experiments are as follows:

  1.  200 mM LiSO4_25 mM Sodium acetate pH 4.5
  2.  15mM Tris-HCl_50mM KCl pH7.5
  3.  1x PBS
  4.  15mM Sodium acetate_100 mM NaCl pH 4.5
  5.  25mM MES_100mM NaCl pH 5.5
  6.  25mM MES_100mM NaCl pH 6.5
  7.  15mM Tris-HCl_100mM NaCl pH 7.5
  8.  15mM Tris-HCl_100mM NaCl pH 8.5

Kindly suggest if the list of buffers is good to work with.  After this, I am planning to perform the aggregation test with each buffer and share it with you. I have a few questions regarding the protein aggregation test, where I need your advice. I have in total 17 mg of protein (19.47 kDa). Would you suggest performing an aggregation test after protein dilution in those particular buffers or after dialyzing protein into these buffers?

Can you please tell me the address where I can ship my samples? When are you planning to perform AUC with these samples and how do you want me to ship them? Furthermore, I would like to know Do you need the buffer in which the protein is dialyzed or the fresh buffers. I will try to send samples accordingly.

Thank you for your time and consideration!

Sincerely,
Jyoti

Postdoctoral Researcher
Gupta Lab
Greehey Children's Cancer Research Institute (GCCRI)
University of Texas Health Science Center at San Antonio
Email: baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>
________________________________
From: Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>>
Sent: Friday, December 15, 2023 12:22 PM
To: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Cc: Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: AUC (sedimentation equilibrium experiments)

Dear Dr. Demeler,
It was very helpful. Thank you!
Will do the needful. I will check do the quick check. I have realized that I forgot to reply about the buffer MBis, it was a typing mistake. It is actually Bis-Tris buffer.

Thank you for your time,
With best regards,
Jyoti

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________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Friday, December 15, 2023 11:48:01 AM
To: Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>>
Cc: Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: AUC (sedimentation equilibrium experiments)

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Hi Jyoti,
the microfuge test is just a quick and easy to perform test for aggregation you can do with a benchtop spectrophotometer, if you are worried about aggregation. It is not essential to do this for an AUC experiment, because AUC does the same thing at much higher resolution. So feel free to skip it, but you may be able to figure out beforehand if it makes sense to test a sample by AUC, unless you test it beforehand.

I hope that clears up your doubts.
Regards, -Borries

On Wed, Dec 13, 2023 at 7:28 AM Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>> wrote:
Dear Dr. Demeler,
Good morning!
Thank you for your comprehensive details related to experiments. I will perform the requisite, measure the OD, make an account, and will upload as well.
There is an important information I would like to share with you, I have never seen any visible precipitate or turbidity in any buffer conditions for this protein, and the 8 different buffer and salt conditions I have mentioned in my previous email have shown good peak when I performed for SEC-MALS. The buffers where it wasn't very well behaving are in very low salt conditions which I haven't sent you the details of yet. I am planning to try these buffers only if these 8 buffer conditions don't work.
I would like to clarify a doubt I have; would you want me to perform aggregation test for these 8 buffers I have mentioned (where I haven't seen any sign of precipitation)? Because in my experience this protein is pretty stable in these buffers and, we only want to see if there is any oligomeric state. However, if it helps in performing AUC, I will be happy to check aggregation in all the buffers.

My sincere appreciation,

With best regards,
Jyoti Baranwal
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Tuesday, December 12, 2023 4:17 PM
To: Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>>
Cc: Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: Re: AUC (sedimentation equilibrium experiments)

*EXTERNAL EMAIL*

Hello Jyoti,
Thanks for the details in your message. It sounds like you may have some issues with aggregation in certain buffers. There is a very simple test you can do if you think this is an issue:

1. measure the absorbance spectrum of your protein from ~ 220-600 nm. If you see non-zero absorbance above 300 nm, you likely have aggregation.
2. put your protein solution in a benchtop microfuge and spin for 3-4 minutes. Carefully aspirate the supernatant and re-measure the absorbance as above. If the absorbance is noticeably lower, then you lost material due to aggregation. Also, make sure the non-zero absorbance >300 nm is gone. That portion is your aggregate.

If the buffers below produce no aggregate, then we can help you measure reversible oligomerization. I am not familiar with MBis, but I do believe acetate buffers absorb below 240 nm, so there we need to know how much. The PBS buffer should not absorb at all in this range if it is clean. The TRIS buffers should be fine as well, as long as you use optical grade TRIS. Also, 50 mM is probably more than you need, so you could go down to 10-15 mM TRIS without losing buffering capability for a low concentration protein. For the acetate buffers I suggest to go to low enough concentration to maintain an absorbance of < 0.3 OD at the lower wavelength where you will want to measure the lower protein concentrations.

It would be good if you could collect buffer absorbance profiles and send them to us for review so we can ascertain optimal conditions.

How much protein do you have available to make these measurements? I would suggest we measure 0.3 OD at 230 nm, and 0.7 OD at 285 nm for each buffer so we can get a good spread of concentrations. Can you please calculate the molar concentrations at these wavelengths and send them to us? You can get a molar extinction coefficient for 280 nm if you upload the sequence into UltraScan's analyte management tool - Yogesh should know how to do that, or we can do it for you.

Please create a LIMS account on our server so we can track this project at this address:
https://uslims.uleth.ca/uslims3_CCH/newaccount.php
Once you created the account, please check your e-mail for an activation link and a temporary password. You can update it after logging in under "Change my info". Next, please submit a project request ("Projects" button in the left bar), and answer questions in the form as best as you know. Leave the experimental design section blank, we will fill this in. Don't forget to include the protein sequence and buffer components. You can also upload PDF documents or images with the absorbance spectra to our LIMS server by clicking on "Supporting Files".

Once this information is uploaded, we will work out a design and we can schedule a zoom call to discuss these experiments further.

Regards, -Borries

I have used a list of buffers to check its oligomeric state of the protein using SEC-MALS. However, there could be possible that salt bridges between the protein is not stable in the SEC-MALS, that's why we aren't able to observe the dimer/oligomeric state of the protein and only get the peak for monomer. We have observed some fraction of protein at higher oligomer when we have lowered the salt concentration in the buffer, but protein seem to be unstable.
We think that AUC would be very helpful to understand the oligomeric state of this protein. Therefore, we want to try different pH, and salt concentration.  We can couple this with multiple concentration of protein, as you have suggested.
I have made a list of buffers we would like to try with this protein (listed below). All the buffer solution has only buffering agent and salt in it. There is no other compound which will hinder absorbance.

  1.  200 mM LiSO4 100 mM Sodium acetate pH 4.5
  2.  50mM Tris-HCl 50mM KCl pH7.5
  3.  1x PBS
  4.  25mM Sodium acetate 100 mM NaCl pH 4.5
  5.  25mM MES 100mM NaCl pH 5.5
  6.  25mM MBis-Tris 100mM NaCl pH 6.5
  7.  25mM Tris-HCl 100mM NaCl pH 7.5
  8.  25mM Tris-HCl 100mM NaCl pH 8.5

On SEC-MALS I have tried two different concentrations 8 mg/ml and 13 mg/ml and 40 ul. To begin with we can start with these buffers and check the oligomeric state if required I have few more buffers to try. We can start with low concentrations of protein and then move to higher concentration. At this stage I can't comment on what concentration we can use for AUC. But what I can say at this point, I have used 200 nM protein on Mass spectrometer it did suggest that it is dimer, but this had a limitation of 30 kDa and because my protein is approx. 19.5 kDa we could not see monomers. Based on the sensitivity of AUC we can decide on the concentration.

Thank you for your time and consideration!

With best regards,

Jyoti Baranwal

Postdoctoral Researcher
Gupta Lab
Greehey Children's Cancer Research Institute (GCCRI)
University of Texas Health Science Center at San Antonio
Email: baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>
________________________________
From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Monday, December 11, 2023 3:34 PM
To: Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>>
Cc: Baranwal, Jyoti <baranwal at uthscsa.edu<mailto:baranwal at uthscsa.edu>>
Subject: Re: AUC (sedimentation equilibrium experiments)

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Hi Yogesh,
thanks for your message! Yes, this sounds very interesting, so we are interested in testing the effect of pH on the kd and the oligomeric state?
I suggest we measure multiple concentrations, perhaps varying 5-20 fold, above and below the putative kd concentration, at each pH you want to test. Any chance we can access wavelengths around 225-230 nm without buffer background? Any buffer component that absorbs should be kept to a minimum concentration so we don't saturate the dynamic range of the detector.

For each sample, we would need about 0.5 ml volume, optical densities at each wavelength should ideally be between 0.3-0.7 OD.

Before we proceed we should have a meeting with our AUC lab members and discuss this research a bit further.

Thanks! -Borries

On Sat, Dec 9, 2023 at 4:51 PM Gupta, Yogesh K <GuptaY at uthscsa.edu<mailto:GuptaY at uthscsa.edu>> wrote:

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Hi Borries,

I hope you are doing well. Jyoti (cc’ed) is a postdoc in my lab who solved the crystal structure of a phosphatase/RNA de-capping enzyme (monomeric molecular mass = ~20 kDa). We observed a dimer in the crystals. Using the sedimentation equilibrium method, we want to check its oligomeric state in solution at different pH  (4.5 – 8.5). Would you be interested in this collaboration? We learned that our AUC in RAB is down, so we didn’t attempt these experiments. We thought you might be interested in this project.

Best,

Yogesh

Yogesh Gupta, Ph.D.
Associate Professor
University of Texas Health Science Center at San Antonio

Department of Biochemistry and Structural Biology

Greehey Children's Cancer Research Institute
8403 Floyd Curl Drive, MC7784

San Antonio, TX, 78229, USA
Phone: 210-562-9064<http://voice.google.com/calls?a=nc,%2B12105629064><http://voice.google.com/calls?a=nc,%2B12105629064>
Gupta Lab<http://ccri.uthscsa.edu/YGupta.html>

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