Protein Sample Preparation and

Quantitation
Kurdianto, Product Manager
February 8, 2021
Agenda

 Introduction
 SureBeads Magnetic Beads System
 Sample Preparation : Purpose and Products
• Protein Extraction
• Protein Clean Up
• Protein Fractionantion
• Protein Depletion
 Protein Quantitation Product
 Market Target dan Positioning
 Introduction

Preparasi sampel protein berkualitas tinggi adalah kunci keberhasilan proses

selanjutnya !!!
 Downstream Applications

 Western Blot
Do not require
 2D Electrophoresis (IEF) active protein
 Mass Spectrophotometry (MS)
 ELISA
 Immunoprecipitation (IP) Require native
conformation
 Reporter Assay
 Purification
 SureBeads Magnetic Beads System

SureBeads are designed for bioseparation

techniques like immunoprecipitation (IP) assays.
SureBeads are superparamagnetic beads with
surface activated hydrophilic polymers and
hemically conjugated Protein A or Protein G that
specifically bind to the Fc region of
immunoglobulins (IgG, IgA, IgM, and IgD)
Feature and Benefits
• Faster IP — no centrifugation
• Easier IP — ergonomically designed, making
washes easy and preventing loss of beads during
pipetting
SureBeads™ Starter Kit Protein A and G (Cat #1614833)
• Less antibody needed — patented surface
chemistry, for optimal antigen binding
• Low cost to go magnetic — priced similarly to
leading agarose beads with the performance of
magnetic beads
• Ready to use – no dilution or aliquoting needed
• Ergonomically designed – easier pipetting
• Better binding – minimize nonspecific binding
• Flexible – available in 16 x 1,5 ml or 2 x 15 ml
format
Immunoprecipitation in five easy steps

Video for more detail : Start

 Sample Preparation : Purpose and Products

Sample
Purpose Products
Prep Step
Break up cells to release their
ReadyPrep Total Protein Extraction Kit,
Extraction contents, get the proteins out of
MicroRotofor Cell Lysis Kits
the cell
Removal of salts, detergents,
nucleic acids, lipids, 2-D Clean Up Kit, Bio-Spin and Micro Bio-
Clean Up
polysaccharides – Substances that Spin Columns
interfere with analysis
ReadyPrep Protein Extraction kits
Reduce sample complexity. This is (specific for cellular locations),
based on a property of the sample ReadyPrep Sequential Extraction Kit,
Fractionation Prep Cell, Arum CEX and AEX mini kits,
such as: cellular location, solubility,
size, charge, pI MicroRotofor

Reduce amount of high abundant

Enrichment ProteoMiner, Arum Serum Protein Mini Kit
protein
Sample Preparation
 Protein Extraction

Goal → Break up cells to release their contents, get the proteins out of the cell

Protein Extraction

Detergent Enzymatic Ready to Use

ReadyPrep™
Denaturating Lysozyme
Protein Extraction

MicroRotofor™ Cell
Non-Denaturating Lysis Kit
 Protein Extraction : Detergent

SDS, 100 g

SDS, 1 kg
Denaturating SDS
SDS Solution 10% (w/v)

SDS Solution 20% (w/v)

Detergent

TritonX-100

Nonionic NP40

Non-Denaturating Tween 20

Zwitterionic CHAPS
Protein Extraction : Enzymatic

 Lysozyme is 129 amino acid residues enzyme

 It is a basic bacteriolytic Protein that hydrolyzes
peptidoglycan, glycosidic bonds and are widely
distributed in nature
 Molecular weight of Lysozyme is an pproximately
14.7 kDa
 It used for bacterial cell lysis
 Protein Extraction : Ready to Use Kit

ReadyPrep Protein Extraction Kit

For animal & plant cells and tissues, yeast, and
bacteria
(Cat #1632086)

MicroRotofor Cell Lysis Kit

Mammalian Cell (Cat#1632141)
Plant Cell (Cat#1632142)
Yeast Cell (Cat#1632143)
Bacterial Cell (Cat#1632144)
 Protein Clean Up / Clarification

Goal → Removal of salts, detergents, nucleic acids, lipids, polysaccharides

Protein Clean Up

ReadyPrep™ 2D Clean Up Kit Micro Bio-Spin Column P-6 Bio-Beads SM-2 Resin
Cat #1632130 Cat #7326221 Cat #1528920

Uses selective precipitation to remove Remove salts and small molecules with Bio-Beads SM-2 Resin is a nonpolar
salts dan ionic contaminants such as a simple 4-minute microcentrifuge spin polystyrene adsorbent. For detergent
detergents, lipids, and phenolic removal ( eg. Triton X-100, NP40,
compounds from protein samples deoxalate, etc)
 Protein Fractionation

Goal → Reduce sample complexity, base on property of the sample

Protein Fractionation

Fractionation by Solubility Fractionation by Cellular Location Fractionation by Charge

ReadyPrep™ Sequential ReadyPrep™ Aurum™ AEX Mini Kit

Extraction Kit Cytoplasmic/Cellular Cat #7326706
Cat #1632100 Cat #1632089

Aurum™ CEX Mini Kit

ReadyPrep™ Extraction Kit ReadyPrep™ Protein
(Soluble/Insoluble) Cat #7326703
Extraction Kit (Membrane )
Cat #1632085
Cat #1632088

ReadyPrep™ Protein
Extraction Kit (Signal)
Cat #1632087
 Protein Depletion
Goal → Reduce amount of high abundant protein, enrich low abundant protein

Protein Depletion

ProteoMiner Protein Enrichment Kits Aurum Affi-Gel Blue Mini Columns Aurum Serum Protein Mini Kit
Cat #1633006 Cat #7326708 Cat #7326701

Use highly diverse bead-based library Effectively remove up to 90% of Enables selective binding and
of combinatorial peptide ligands to albumin from serum or plasma simultaneous removal of both albumin
decrease high abundance protein samples by affinity chromatography and IgG from serum or plasma samples
concentration (resin-based) (resin-based)
 Protein Quantitation : Assay Principle

Bradford protein assay is based on the

absorbance shift of the dye Coomassie
brilliant blue G-250

Lowry protein assay is based on the reaction

of copper ions (Cu+) ions produced by the
oxidation of peptide bonds with Folin–
Ciocalteu reagent
Sample Quantitation

Protein Standard
(Bovine Serum Albumin & BɣGlobulin)

Pre-diluted Protein Standard, 7 vilas

(0.125–2.0 mg/ml)
 Target Market dan Positioning

Target Market

 Protein research labs

 Biopharma R&D and QC
 Medical faculty
 Hospital laboratory
 Basic/advance university lab that doing
biomarker discovery, antibody isolation and
purification
Thank You