[Demelerlab] AUC (sedimentation equilibrium experiments)

Tue Dec 12 15:17:50 MST 2023

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 <baranwal at uthscsa.edu>*
> ------------------------------
> *From:* Borries Demeler <demeler at gmail.com>
> *Sent:* Monday, December 11, 2023 3:34 PM
> *To:* Gupta, Yogesh K <GuptaY at uthscsa.edu>
> *Cc:* Baranwal, Jyoti <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> 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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