Outline o Online purification o Why and How to do SEC-SAXS (+SLS)? o - - PowerPoint PPT Presentation

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Outline o Online purification o Why and How to do SEC-SAXS (+SLS)? o - - PowerPoint PPT Presentation

Feb 19, 2023 351 likes •683 views

Outline o Online purification o Why and How to do SEC-SAXS (+SLS)? o Why not to do SEC-SAXS? o Current/ Future Developments Recources https://padlet.com/melissagraewert/P12_UM2020 password:um2020 SEC-SAXS Paper Online Lecture Course

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Outline

  • Online purification
  • Why and How to do SEC-SAXS (+SLS)?
  • Why not to do SEC-SAXS?
  • Current/ Future Developments
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SLIDE 2

Recources

https://padlet.com/melissagraewert/P12_UM2020 password:um2020 Online Lecture Course

  • Lecture 3 – Sample and

Buffer Preparation

  • Lecture 6 - Mixtures

SEC-SAXS Paper

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SLIDE 3

I(s) s sample buffer subtracted curve Shape determination Flexible systems Missing fragments Structure validation Rigid body modelling

Why SEC-SAXS(/MALLS)?

monodisperse

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I(s) s sample buffer subtracted curve Shape determination Flexible systems Missing fragments Structure validation Rigid body modelling

Why SEC-SAXS(/MALLS)?

polydisperse

1/15

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SLIDE 5

I(s) s

sample buffer subtracted curve Shape determination Flexible systems Missing fragments Structure validation Rigid body modelling

Why SEC-SAXS(/MALLS)?

Size Exclusion

1/15

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SLIDE 6

SAXS modes (by visit)

  • 2019

EMBL Beamline P12 at Petra III: SEC-SAXS

SEC-SAXS has become a standard set-up over the last years

2/15

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SLIDE 7

EMBL Beamline P12 at Petra III: SEC-SAXS

SEC-SAXS has become a standard set-up over the last years

Graewert, M.A. and Svergun, D.I. (2020) Biochem (Lond) 42(1), 36-42

3/15

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How to do SEC-SAXS(/MALLS) at P12?

Check Box on the Application Short justification required in the proposal text  See talk von Maria Vanoni Why: Your Local Contact will be aware and prepared If circumstances change: And you would like to use SEC-SAXS  Communicate with us. (we can find a way)

Apply Prepare Preform Analyse

4/15

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SLIDE 9

How to do SEC-SAXS(/MALLS) at P12?

Communicate in advance:

  • What Column(s) – check availability/

suitability

  • What Buffer(s) – identify issues (Zn2+,

Phenol, DTT,…)

  • How many runs – check practicality
  • Any special requests – no surprises

Bring / Send

  • Sufficient Buffer
  • Sufficient Sample
  • Time
  • (Column)

Apply Prepare Perform Analyse

5/15

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SLIDE 10

How to do SEC-SAXS(/MALLS) at P12?

Communicate in advance:

  • What Column(s)
  • What Buffer(s)
  • How many runs
  • Any special requests

Bring / Send

  • Sufficient Buffer
  • best STOCK (dilute, filter, degas) on-site
  • send additives such as DTT (or confirm

availability)

  • Sufficient Sample
  • for 10/300 column, 50-120 ul (>5mg/ml)
  • for 5/150 column, 10-50 ul (>3mg/ml)
  • Time
  • for 10/300 column, 50 + 10 min per run
  • for 5/150 column, 15 + 5 min pre run

+ PREP time

  • (Column)

Apply Prepare Perform Analyse

5/15

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SLIDE 11

Prepare – WHAT COLUMN

6/15

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SLIDE 12

Prepare – WHAT COLUMN Balance Concentration & Resolution

Low concentration  small column High concentration + no “high resolution” required  small column High concentration + “high resolution” required  large column Other factors: - parallel MALLS

  • sample stability/ equilibrium

(- time limitations)

6/15

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How to do SEC-SAXS(/MALLS) at P12?

CHECK:

Apply Prepare Perform Analyse

7/15

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How to do SEC-SAXS(/MALLS) at P12?

Mail-in:

Do not forget clear instructions such as

  • Thaw slowly/quickly
  • Anything to be added
  • Removal of potential aggregates through

Centrifugation or filter

  • Gives us warnings such as:
  • “sample aggregates after 2-3 hours”
  • “peaks might not be base-line

separated  less volume should then be injected”

  • “we normally expect pressure build

up”

Apply Prepare Perform Analyse

8/15

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How to do SEC-SAXS(/MALLS) at P12?

On-site:

  • Introduction to the system
  • HPLC training
  • Words of caution
  • Introduction to Becquerel settings
  • 2-3 trial runs
  • We trust you with the system
  • Cleaning up the system
  • Column washed
  • System in water
  • Disconnected from the bealmine

Apply Prepare Perform Analyse

9/15

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How to do SEC-SAXS(/MALLS) at P12?

On-site:

  • Introduction to the system
  • HPLC training (let us know if you

do not have much experience)

  • Words of caution (take note for

late shift)

  • Introduction to Becquerel settings

Now easy to start/switch, automated cleaning

  • 2-3 trial runs
  • We trust you with the system

More support required with MALLS

  • Cleaning up the system (also when

you are tired)

  • Column washed/System in water
  • Disconnected from the beamline

Apply Prepare Perform Analyse

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Perform !!!! Leaks, Buffer Fillings, Pressure Issues, …

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How to do SEC-SAXS(/MALLS) at P12?

Receive Data:

  • Automatically receive:
  • Data files
  • If required, request 2D images
  • Automatic Chromixs/SASFLOW

evaluation

  • Check data as soon as possible and

contact for follow-up questions

Apply Prepare Perform Analyse

10/15

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SLIDE 19

Analyse

Graewert et al (2020)

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SLIDE 20

Analyse

Graewert et al (2020)

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Why SEC-SAXS(/MALLS)?

  • Analysis of individual components
  • No issues with buffer matching
  • Concentration series on the “fly”
  • Addition of further detectors:

independent estimate of MW, assessment of re-mixing/re-

  • ligomerization, additional information

for membrane proteins

11/15

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column interactions

Why NOT TO DO SEC - SAXS(/MALLS)?

12/15

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A B

  • Nice peak
  • Removal from unbound substance
  • Return to background
  • Good data!

SEC-SAXS

Why NOT TO DO SEC - SAXS(/MALLS)?

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A B

SEC-SAXS

Why NOT TO DO SEC - SAXS(/MALLS)?

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column interactions radiation damage

Why NOT TO DO SEC - SAXS(/MALLS)?

13/15

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SEC-SAXS

30/35

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column interactions radiation damage dilution effect column resolution

Why NOT TO DO SEC - SAXS(/MALLS)?

14/15

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SEC-SAXS

  • There are some limits!

“Hmmm, I don’t know which peak it is?”

Why NOT TO DO SEC - SAXS(/MALLS)?

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1.Trip:

  • 2. Trip:
  • There are some limits!

Why NOT TO DO SEC - SAXS(/MALLS)?

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When performing SEC-SAXS(+MALLS)? Remember

“ideal sample”

  • Pre-analysis of sample is very important; optimize

SEC conditions not quite pure sample

  • SEC-SAXS is analytical! Not preparative!

radiation damage can be an issue

  • Measure batch sample as well, add scavengers

sample stability, low affinity complexes

  • sample can be altered with column interaction

15/15

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New SAXS tools for the characterization of Biologics Proof of principle: Analysis of IG1 domains* after Papain digestion AXS/MALLS

*these are similar in size and low in concentration (classic SAXS/SEC-SAXS not possible) FAB; Χ² = 1.6 FC; Χ² = 1.8

Contact us if you are interested in performing Online IEC

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SLIDE 32

EMBL Hamburg: The SAXS community SAXS group Instrumentation team User Office Funding: Aknowledgements

Beamline: Clement Martin Andrey Software: Al, Dima, Daniel, Nelly HPLC: Cy Stefano Tobi Haydyn IEC: Taja