Sample and buffer preparation Melissa Grwert EMBL Hamburg Biology - PowerPoint PPT Presentation

Solution Scattering from Biological Macromolecules Sample and buffer preparation Melissa Gräwert EMBL Hamburg

 Biology (Dipl.) in Heidelberg (Biochemistry/physics)  PhD in Stuttgart (Biophysics)  Postdoc in Munich (MX)  Joined the SAXS group

 Planning an Experiment  Assessing Sample suitability  Preparing an Experiment  Best sample  Best buffer  Performing an Experiment  Trouble shooting at the beamline: 5 test cases Overview

 The SAXS (small angle X-ray scattering) Experiment I(s) X-ray beam 2 θ s s PLANNING THE EXPERIMENT 1/35

 The SAXS (small angle X-ray scattering) Experiment I(s) X-ray beam s PLANNING THE EXPERIMENT 1/35

 Sample requirements  Amount: 5 μ l (30 μ l); 40 μ l per sample  Concentration:  Buffer:  Sample quality: PLANNING THE EXPERIMENT 2/35

 Sample requirements  Amount: 5 μ l; 40 μ l per sample  Concentration: more particles  stronger signal  Buffer  Sample quality I(s) I(0) ≈ N (∆ρ V)² 7.5 mg/ml 3.7 mg/ml 15 mg/ml s PLANNING THE EXPERIMENT 2/35

 Sample requirements  Amount: 5 μ l; 40 μ l per sample  Concentration: dependent on MW (100 ~ MW x c)  Buffer  Sample quality I(s) I(0) ≈ N (∆ρ V)² ~ 70 kD < 20 kD > 400 kD s PLANNING THE EXPERIMENT 2/35

 Sample requirements  Amount: 5 μ l; 40 μ l per sample  Concentration: dependent on MW  Buffer: as less additives as possible  Sample quality I(0) ≈ N ( ∆ρ V)² No additives small amount high amount PLANNING THE EXPERIMENT 2/35

 Sample requirements  Amount: 5 μ l; 40 μ l per sample  Concentration: dependent on MW  Buffer: as less additives as possible  Sample quality Foldon vs Proteasome PLANNING THE EXPERIMENT 2/35

 Planning an Experiment  Assessing Sample suitability: ~0.1 - 0.5mg of purified sample  Preparing an Experiment  Best sample  Best buffer  Performing an Experiment  Trouble shooting at the beamline: 5 test cases Overview

 Sample preparation strategies  Different modes of access  on-site visits (48 hours)  mail-in operation PREPARING THE EXPERIMENT 3/35

 Sample preparation strategies  Sample stable at high concentrations UV Concentrate Dialyze (Store/Ship) Filter Conc. determination SAXS from dilutions & buffer  Sample not stable at high concentrations UV Dialyze (Store/Ship) Filter Step-wise Conc. determination SAXS from different conc. concentration centration steps & buffer PREPARING THE EXPERIMENT 4/35

 Sample Characterization nativeMS Analytical ultracentrifugation percentage m/z) Native Gel Size Exclusion Dynamic Light Static Light Electrophoresis Chromatography Scattering Scattering PREPARING THE EXPERIMENT 5/35

 Buffer Preparation  Method of choice: Dialysis - diffusion coefficients - temperature - time - concentration of species - sample volume - dialysate (buffer) volume (100:1) - number of dialysate changes (2-3) - membrane surface area - membrane thickness - molecular charges - dialysate agitation (stirring)  standard protocol: 16 to 24 hours PREPARING THE EXPERIMENT 6/35

 Buffer Preparation  Method of choice: Dialysis Tubing for ~2mL to 100mL samples Cassettes for ~0.5mL to 70mL Cup Devices for ~10μL to 2mL  Alternative methods Spiking Diafiltration Desalting/SEC column PREPARING THE EXPERIMENT 6/35

 Batch mode or SEC-SAXS mode PREPARING THE EXPERIMENT 7/35

SEC-SAXS /SLS: Alternative strategy to study (moderatly) polydisperse samples How is SEC-SAXS done • required sample amounts: at least 50ul, >5mg/ml • sufficient buffer • optimize your SEC run • if possible collect batch sample as well • check for radiation damage, add 1-3% glycerol (if feasible) PREPARING THE EXPERIMENT 8/35

 For SEC-SAXS mode, k eep in mind:  Moderately polydisperse samples  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  More during lecture 6 PREPARING THE EXPERIMENT 8/35

 Case 1: the Relaxed Scientist  Sample:Calmodulin; 100 ul 6.5 mg/ml (UV-Vis)  Question: confirm monomeric state (16.8 kD)  Result: I(0)  22 kD  (30% higher) PERFORMING THE EXPERIMENT 9/35

 Case 1: the Relaxed Scientist  Sample:Calmodulin; 100 ul 6.5 mg/ml (UV-Vis)  Question: confirm monomeric state of protein  Result: MW expected = 16.8 kD MW I(0) = 22 kD (30% higher)  Explanation: ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEV DADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVM TNLGEKLTDEEVDEMIREANIDGDGQVNYEEFVQMMTAK PERFORMING THE EXPERIMENT 9/35

 Case 1: the Relaxed Scientist  Sample:Calmodulin; 100 ul 6.5 mg/ml (UV-Vis)  Question: confirm monomeric state of protein  Result: MW expected = 16.8 kD MW I(0) = 22 kD (30% higher)  Explanation: unsuitable method for determining c ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEV DADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVM  𝜻 = 𝟏. 𝟐𝟖 TNLGEKLTDEEVDEMIREANIDGDGQVNYEEFVQMMTAK protparam: Experience shows that this (no Trp) could result in more than 10% error in the computed extinction coefficient PERFORMING THE EXPERIMENT 9/35

Method Principle Advantages Limitations Lowry Assay - Buiret chormophore - Relative sensitive: -interfering compounds such (copper ion complex with - 1 to 100 ug as detergents, carbohydrates, glycerol, Tris , EDTA… amide bonds) - Cu +I, Tyr, Trp reduce Folin- -- content of Tyr, Trp Ciocalteu reagent (660 nm) - time consuming BCA Assay - protein backbone chelates -less sensitive to the - Cysteine rich samples (temp) Cu 2+ ions and reduces them bicinchoninic acid types of amino acids - reducing agents (DTT, 2-ME) to Cu 1+ which shifts color of in the protein -time consuming dye (562 nm) - suitable for most detergents & denaturants Bradford Assay - Color shift of Coomassie -simple, rapid, -- content of Arg (eg. histones) brilliant blue G-250 dye cheap, sensitive -- non linear curve (absorbance upon binding arg and - micro: 1-20 ug of free dye) aromatic residues - macro: 20-100 ug - Choice of standard, pH - “sticky proteins” precipitate - copes with reducing agents UV( 280 nm) Ultraviolet absorbance - quick - sequence dependent according to - sample recovery - protein complexes, mixtures Beer’s law, A~c ε -- sensitive to pH and ions Differential index of refraction according - total/ pure protein -Magic number Refractometry to Snell's law - quick -Temperature sensitive - sample recovery PERFORMING THE EXPERIMENT 10/35

dual cell, deflection design Rudolph Research Analytical J357 refractometer PERFORMING THE EXPERIMENT 11/35

 Assay choice  Goal: accuracy (compared to precision)  Consider sample composition (sequence)  Consider buffer composition (pH, additives)  Consider a precipitation step to remove buffer  Sample volume (High-through put)  Protocol for choosing suitable method, eg. Olson, Markwell; Curr Protoc Protein Sci. 2007(3) PERFORMING THE EXPERIMENT 12/35

 Case 1: the Relaxed Scientist  Question: confirm monomeric state of protein  Result: MW expected = 16.8 kD MW I(0) = 22 kD (30% higher)  Explanation: unsuitable method for determining c  Solution: use different method  c= 8.6 mg/ml confirm with other methods  MW: 16.6 PERFORMING THE EXPERIMENT 13/35

 Case 1: the Relaxed Scientist  Sample:Calmodulin; 100 ul 6.5 mg/ml (UV-Vis)  Question: confirm monomeric state (16.8 kD)  Result: Porod volume  28.14 nm 3  17 kD PERFORMING THE EXPERIMENT 13/35

 Case 2: the Lazy Scientist  Sample: 11 mg/ml Lysozyme in 30 and 90 mM NaCl  Question: effect of adding salt  Conclusion increase in NaCl, unfavorable for the protein PERFORMING THE EXPERIMENT 14/35

 Case 2: the Lazy Scientist  Sample: 11 mg/ml Lysozyme in 30 and 90 mM NaCl  Question: effect of adding salt  Result: Rg 30mM = 1.0 nm Rg 90mM = 1.2 nm  Rg expected = 1.4 nm PERFORMING THE EXPERIMENT 14/35

PERFORMING THE EXPERIMENT 15/35

11 mg/ml 5.5 mg/ml 2.75 mg/ml PERFORMING THE EXPERIMENT 16/35

11 mg/ml 5.5 mg/ml 2.75 mg/ml PERFORMING THE EXPERIMENT 16/35

 Case 2: the Lazy Scientist  Sample: 11 mg/ml Lysozyme in 30 and 90 mM NaCl  Question: effect of addition of salt  Result: RG expected = 1.4 nm = 1.0 → 1.4 nm RG 30mM = 1.2 → 1.4 nm RG 90mM  Explanation: concentration effects  Solution: measure different concentrations PERFORMING THE EXPERIMENT 17/35

 Case 3: the Ambitious (Hasty) Scientist  Sample: well characterized mutants, different ligands  Question: understanding the binding mechanism PERFORMING THE EXPERIMENT 18/35

 Case 3: the Ambitious (Hasty) Scientist  Sample: well characterized mutants, different ligands  Question: understanding the binding mechanism  Result: look at automated pipeline PERFORMING THE EXPERIMENT 19/35

PERFORMING THE EXPERIMENT 20/35