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![Purification of IgG from CHO cell culture
using MonoSpin ProA
15
SDS page
1. MW
2. CHO cell culture
3. Flow through
4. Washing
5. Elution
6. Regeneration
Sample amount of IgG in CHO cell culture [mg]
RecoveryofIgG[mg]
High selectivity and good recovery.](https://image.slidesharecdn.com/lifescienceproductsgls-200122105548/75/Life-science-products-of-GL-Sciences-15-2048.jpg)
Uploaded byGeert Alkema
Life science products of GL Sciences
The document describes various life science products for sample preparation and analysis, including: 1) MonoSpin SPE columns for solid phase extraction of small volume samples. 2) MonoTip pipette tip SPE for efficient concentration and purification of peptide and protein samples. 3) Titansphere Phos-TiO kits for selective enrichment of phosphopeptides from protein digests. 4) MonoSpin ProA and ProG kits for rapid antibody purification using monolithic silica spin columns. 5) MonoCap high resolution LC columns for high efficiency peptide and protein separation.
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- 1. Life Science products Dec6,2019 1
- 2. MonoSpin Series SPE Spincentrifuge columns 2
- 3. Small volume SPE 3 10g/60 mL 5 g/20 mL 2 g/12 mL 1 g/6 mL 200 mg/3 mL 50 mg/1 mL 500 mg Blue: Capacity 250 mg 100 mg 50 mg 10 mg 2.5 mg 24 mL Violet: min. Elution volume 12 mL 4.8 mL 2.4 mL 500 μL 125 μL 500 μg 50 μL Scale down
- 4. MonoSpin 4 - is aspin column packed with monolithic silica for solid phase extraction. - can be used for sample pretreatment with the centrifuge method. - is useful and effective for sample pretreatment when the biological sample volume is small. MonoSpin Monolithic silica disk is fixed into a mini spin column.
- 5. Monolithic silica 5 Monolithic silicagel is composed by ethyl silicate, and it has uniform three-dimensional structure. It has several advantages such as highly effective for small volume purification and concentration. Sample solution flows through the through-pore, and it is separated while a sample components and functional ligands cause distribution on a silica gel frame. Compared with the space of the silica gel particle, the space of the monolith silica gel have more high uniformity and selectivity. Fig 1. The novel mini spin column. (MonoSpin) Fig 2. Monolihic silica disk. Diameter 4.2 mm, Thickness 1.5 mm Fig 3. Electron microscope photograph. Fig 4. Showing through pore and meso pore. Silica gel （frame） Through pore Meso pore
- 6. Procedure for MonoSpin 6 1.Conditioning (equilibrating) 2. Sample Absorption 3. Rinsing 4. Elution 5. Purified sample Spin Spin Spin Spin : 10,000 rpm for 30secSpin Put a spin column on a centrifuge tube. Each solution are flushed into a column with a centrifuge. Only 10 minutes to complete operation!
- 7. MonoSpin series lineup 7 MonoSpin C18 MonoSpin SAX MonoSpin NH2 MonoSpinTiO MonoSpin Amide (CH2 )3 N + (CH3 )3 Cl (CH2 )17 CH3 MonoSpin C18-AX MonoSpin Trypsin MonoSpin C18-CX MonoSpin PBA MonoSpin SCX MonoSpin CBAMonoSpin Ph
- 8. Desalting of trypticdigested(peptides) 8 0 10 20 Time (min) 0.20.40.60.8 mV 0 10 20 Time (min) 0.20.40.60.8 mV Denaturant & salts 0 10 20 Time (min) 0.1600.1700.1800.1900.2000.210 mV 0 10 20 Time (min) 0.1600.1700.1800.1900.2000.210 mV Enlarged view HPLC Condition Sample: digested BSA 2 μL Column: Inertsil ODS-3 (3 μm 2.1 x 150 mm) Mobile fase A: Water(0.1 %TFA) B: ACN( 0.1 % TFA) Grad A:B=90:10 – 5 min 90:10 – 20 min - 50:50 Detection UV 210 nm Flow rate 0.2 mL/min Temp 40 ℃ Before After MonoSpin C18 can be used for desalting of peptides sample.
- 9. Purification of catecholamine 9 010 20 Time (min) 100200 nA 0 10 20 Time (min) 100200 nA 0 10 20 Time (min) 100200 nA ＜LC Condition＞ Column: Inertsil ODS-3 (150 X 2.1 mm I.D.) Elution: 50 mM phosphate buffer(pH5.6) 50 mg/L EDTA 600 mg/L IPCC-008-10 % Methanol Flow rate=300 mL/min Injection: 5 mL Detector: ECD703 plus Diamond electrode +800 mV Ag/AgCl Sample 1:Noradrenaline, 2:Adrenalin, 3:DHBA, 4:Dopamine Temp. 35 ℃ Purified with MonoSpin PBA (Sample load : 500 μL, Elution : 50 μL) Purified with MonoSpin PBA (Sample load : 500 μL, Elution : 500μL) 1 2 3 4 1 2 3 4 Urine
- 10. Strategy of Batchtrypsin digestion 10 Proteins Trypsin Peptides + Trypsin ＋ Digestion 10hr Proteins Identification LC/MS/MS Proteins : Trypsin = 50 : 1 Proteins Trypsin Peptides +Trypsin ＋ Digestion Few min. =Trypsin Identification LC/MS/MS Proteins : Trypsin = 50 : 50 General method No good good
- 11. Strategy of MonoSpinTrypsin digestion 11 Proteins MonoSpin Trypsin Peptides + Trypsin Protein Identification LC/MS/MS High density Trypsin immobilized Silica monotlih Digestion Few min.
- 12. Batch digestion vsMonoSpin Trypsin 12 0 10 20 Time (min) 0.100.200.30 mV 0 10 20 Time (min) 0.100.200.30 mV Batch digestion 36 ℃ 10 hr MonoSpin Trypsin: Room temp. 10 min centrifuge HPLC Condition Sample: digested BSA 2 μL Column: Inertsil ODS-3 (3 μm 2.1 x 150 mm) Mobile fase A: Water(0.1 % HCOOH) B: ACN( 0.1 % HCOOH) Grad A:B=90:10-20 min-50:50 Detection UV 210 nm Flow rate 0.2 mL/min Temp 40 ℃
- 13. MonoSpin ProA、ProG Ultra fastantibody purification kit using monolithic silica 13
- 14. MonoSpin ProA/ProG 14 The MonoSpinProA/ProG is a monolithic spin column chemically bonded with affinity ligand for purification of antibody, which is ideal for high speed purification. 〇2 type of ligand (ProteinA, ProteinG) 〇Total operation time 10 min 〇Spin column, 96 Well Plate, Large spin column format 1. Conditioning 2. sample 3. Washing 4. Elution 5. Purified IgG Spin Spin Spin Spin Total operation time < 10 min Spin column 96 Well Plate Large spin column
- 15. Purification of IgGfrom CHO cell culture using MonoSpin ProA 15 SDS page 1. MW 2. CHO cell culture 3. Flow through 4. Washing 5. Elution 6. Regeneration Sample amount of IgG in CHO cell culture [mg] RecoveryofIgG[mg] High selectivity and good recovery.
- 16. Recovery with smallelution volume 16 MonoSpin ProA show the IgG recovery more than 90 % with small elution volume(<100 μL). Other kits require a large sample volume for effective elution. Fraction Vol. MonoSpin ProA Elution vol. 100 μL Recovery >90 % ※ * Not available Brand G 400 μL: 70 % Bland T 400 μL: 65 % 200 μL, 100 μL : Not available Recovery（%） These kits require a large sample volume for elution.
- 17. Vs. Agarose media 17 Agarosemedia Competitor GE healthcare Hitrap Spin ProA
- 18. High capacity spincolumn MonoSpin L ProA、ProG 18 ProteinA, ProteinG 8 mL ProA human IgG 16 mg ProG human IgG 12 mg Lingand Capacity Sample volumne Specification NEW
- 19. Easy Operation bycentrifuge 19 1. Binding buffer 5 mL 2. Sample 8 mL (filtered through a 0.2 µm filter to remove particulates) 3. Washing buffer 5 mL 4. Elution buffer 5 mL Each centrifuge 1500xg, 2 min Column setting
- 20. MonoTip Pipette tip SPEusing monolithic silica disk 20
- 21. MonoTip pipette tipSPE 21 Features •Easy-to-Operate • With MonoTip C18, samples are processed by pipetting. The operation is easier and less time consuming than using conventional SPE methods •High efficiency with a wide range of molecular weight • Continuous through-pore is the structural advantage of silica monolith, which enables MonoTip C18 to offer high efficiency. MonoTip C18 is available for peptide and protein samples with the concentration range from pmol to nmol • Adapted to Peptide-Protein Samples with a Molecular Weight of Up to 40,000 in the Sample Concentration pico mol to nano molar order MonoTip Monolithic silica disk
- 22. How to useMonoTip 22
- 23. MonoTip C18 applications 23 010 20 Time (min) 0.000.02 Volts ＜High recovery＞ ＜Conditions> Column: Inertsil WP300 C18 (150 X 2.1mm I.D.) Elution: A: water (0.1%TFA) B: Acetnitrile/water=90/10 (0.1%TFA) A/B = 80/20-(20min)-40/60 Flow rate=0.3mL/min injection: 5uL Detector: UV280nm Sample: Cytochrom c 60μg in 100μL After Recovery 95% ＜Effective sample concentration＞ 0 10 20 Time (min) 0.00.2 Volts ＜Conditions＞ Column: Inertsil WP300 C18 (150 X 2.1mm I.D.) Elution: A: water (0.1%TFA) B: Acetnitrile/water=90/10 (0.1%TFA) A/B = 90/10-(20min)-40/60 Flow rate=0.3mL/min Injection= 5μL Detector: UV2１0nm Sample: Beta-casein 10μｇ trypsin digested in 100μL Elution volume 20 μL 0 10 20 Time (min) 0.00.2 Volts 0 10 20 Time (min) 0.000.02 Volts Before
- 24. Selective enrichment of phosphopeptides Proteomicsrelated products 24
- 25. Purification of phospho-peptides 25 1mg / 10 mL 3 mg / 200 mL 50 mg/ 3 mL (A) (C) (D) Figure Product description (a) Titanium dioxide particle (b) Titansphere PHOS-TiO Kit (c) Tip Column (d) Cartridge Column The Kit is used to enrich phosphopeptide from digested protein or peptide. (B) Titansphere Phos-TiO Kit contains titansphere material in a tip-column designed to use with centrifuge.
- 26. Phosphopeptide purification procedure 26 Thetotal operation of "Titansphere Phos-TiO Kit" is only four steps, conditioning / adsorption / washing / elution. The total operation time is only 40 minutes.
- 27. Comparison of therecovery and selectivity between the commercial phosphopeptide enrichment kits. 27 0 100 200 300 400 500 600 Competitor D (IMAC) Competitor C (TiO2) Competitor B (TiO2) Competitor A (TiO2) Titansphere Phos-TiO Kit Relative Peak Area(%) Phosphopeptides non-phosphopeptides Number of Identified Peptides (Selectivity) (phospho-/non-) 97.4 % ( 12 / 1 ) 71.4 % ( 12 / 7 ) 25.1 % ( 4 / 5 ) 71.5 % ( 9 / 7 ) 23.5 % ( 5 / 9 ) The following chart shows recovery and selectivity of four commercially available phosphopeptide purification kits and the GL Sciences’ Phos-TiO system. Note how Phos-TiO shows virtually zero non-specific adsorption without sacrificing capacity and recovery.
- 28. Titansphere Phos-Tio MPKit Efficiently Enrich BOTH singly and Multiply Phosphorylated peptide 28
- 29. Titansphere Phos-Tio MPKit 29 Features High Recovery of not only Singly, but also for Multiply Phosphorylated Peptides. All Operation is done using an Easy to use centrifuge Efficiently Enrich BOTH singly and Multiply Phosphorylated peptide
- 30. IMAC and Phos-TioStratagy 30 Weak interaction Suitable for Multiple phosphopeptide Strong interaction Suitable for Singly Phosphopeptide
- 31. Comparison of Recoveryof BOTH Singly and Multiply Phosphorylated Peptieds 31
- 32.
- 33. Fractionation of Peptides 33 Trypticdigests Step-wise peptide elution from SDB-SCX StageTip Dry up LC-MS/MS
- 34. Comparison of TraditionalGradient Elution vs TFA Gradient Elution 34
- 35. Comparison of Numberof Quantified peptides 35
- 36. Desalting spin columnfor small volume sample 36 GL-Tip GC GL-Tip SDB Specifications Product GL-Tip SDB Solid Phase Styrene Divinylbenzene Sample Try(PO3H2)-AngiotensinⅡ Tip volume 200 μL Capacity 60 μg Product GL-Tip GC Solid Phase Graphite carbon Sample Gly-Gly-Tyr-Arg Tip volume 200 μL Capacity 30 μg GL-Tip GC • Purification tool for hydrophilic peptides Micropipette used for Desalting of TiO2-Enriched Samples Prior to LC/MS
- 37. Recovery of peptidesin several desalting tools. 37 0 10 20 T ime (min) 0100200 mAU 0 10 20 T ime (min) 0100200 mAU 0 10 20 T ime (min) 0100200 mAU 0 10 20 T ime (min) 0100200 mAU Protein Digested Without desalting Zip-Tip C18 Low recovery GL-Tip SDB High recovery GL-Tip GC High recovery for Hydrophilic peptides Hydrophilic peptides
- 38. High Efficiency NanoLC column MonoCap Highresolution 2000 38 ‘High efficiency column ‘ (large N) instead of ‘high speed column’ (large N/t0) Column Length : 2000 mm N=200,000 MonoCap High Resolution 2000 MonoCap HighResolution 2000 is a 2 meter length monolithic silica capillary column which is designed for identifying extremely high number of peptides/proteins for proteome research via LC-MS/MS.
- 39. Performance of MonoCapHigh resolution 2000 ~High column efficiency~ 39
- 40. Recommended Protocols foromix analysis 40 EV Second, MonoSpin ProA MonoSpin Trypsin HP Phos-Tio GL-Tip SDB MonoCap HighResolution 2000 MonoCap HILLIC UP GL-Tip SDB-SCX MonoCap (Column length 150 mm)