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Protein Biology


Protein biology is one of the oldest pillars in the study of cells. Prior to identification of DNA and genes, proteins were recognized as the workhorses of the cells: critical for its functioning. This was also possible by the ease of isolating proteins in abundance from cells, tissues and other sources. Thus, protein isolation, chromatography, structure and function elucidation have been well established techniques.

Solutions from

Protein expression (in a test tube) however is a more recent addition. This topic is covered under Cell culture and Transfection workflows.

  • Common Steps in a typical protein workflow
  • Protein Identification & Detection Techniques
  • Purified peptides, recombinant proteins and tagged proteins
  • Common Equipment

Common steps in a typical protein workflow

Protein Extraction

Protein extraction is the oft used method for isolation of proteins. A critical requirement of this step is efficient cell lysis and separation of organelles if specific proteins are to be extracted from these. The methods used for extraction depend on the type of tissue, samples and organelle needed to the separated. For more information visit: Synopsis.

Factors to consider in this step are type of cell disruption needed: Plant cells have cellular walls which are difficult to break, bacterial cell walls can be sonicated, tissues such as bones and others tough tissues benefit from freeze-fracturing. Multiple options for homogenization and cell disruption are available from OMNI International.

Secondly, use of detergents, protease inhibitors, protein stabilization agents are common to ensure protein function. This is necessary for applications such as ELISA and crystallography which require both function and structure of the proteins to be maintained. Furthermore, clinical samples such as blood, urine etc., contain inhibitors and proteases- hence choice of buffers and steps in the workflow should be made keeping the above parameters in mind.

An often used technique to purify protein in large abundance is to clone it in vectors and express it in suitable cells and cell lines.

Cell lysis solutions for these are:

  • Bacterial Cell Lysis
  • Insect Cell Lysis
  • Yeast Cell Lysis
  • Plant Cell Lysis

Solutions for culture mammalian cells and human tissues are: Protein Extraction Solutions

Additional resources on protein extraction
Technical guide and handbook

Popular products for Protein Extraction

Product Types of Samples Assay Compatibility Compatible Applications
T-PER  Tissue Protein Extraction Reagent
Cat# 78510
Heart, liver, kidney, lung, spleen BCA, Coomassie Plus, Pierce 660, Detergent Compatible Bradford IP, Western blots, ELISA, EMSA, purification, kinase assays, activity assays & amine reactive labeling
N-PER  Neuronal Protein Extraction Reagent
Cat# 87792
Brain tissue and primary neurons BCA, Pierce 660, Detergent Compatible Bradford IP, pulldowns, Western blots, ELISA, enzyme assays & amine reactive labeling
M-PER Mammalian Protein Extraction Reagent
Cat# 78501
Cultured Mammalian and primary cells BCA, Coomassie Plus, Pierce 660, Detergent Compatible Bradford IP, Western blots, ELISA, EMSA, reporter assays, purification, activity assays & amine reactive labeling
RIPA Lysis Buffer
Cat# 89900
Cultured Mammalian cells BCA, Detergent Compatible Bradford (dilute 1:3) Western blots, ELISA & purification
IP Lysis Buffer
Cat# 87787
Cultured Mammalian cells BCA, Pierce Detergent Compatible Bradford Western blots, ELISA,
IP & reporter assays

Additional Solutions for Protein Extraction

Protease Inhibitors Catalogue Number
Pierce™ Protease Inhibitor Mini Tablets, EDTA-free A32955
AEBSF Protease Inhibitor 78431
E-64 Protease Inhibitor 78434
Bestatin Protease Inhibitor 78433
Aprotinin Protease Inhibitor 78432
Halt™ Protease Inhibitor Cocktail, EDTA-Free (100X) 78437
Pepstatin A Protease Inhibitor 78436
Pierce Protease and Phosphatase Inhibitor Mini Tablets A32959
Pierce Protease and Phosphatase Inhibitor Mini Tablets,EDTA-free A32961
Detergents Catalogue Number
Tween™ 80 Surfact-Amps™ Detergent Solution 28328, 28329
28320
Triton™ X-100 Surfact-Amps™ Detergent Solution 28314, 28332
NP-40 Surfact-Amps™ Detergent Solution 28324, 85124, 85125
Brij™-35 Surfact-Amps™ Detergent Solution 28316, 85117, 85118

Protein Isolation & Purification

Various options are available for protein purification. Notable amongst them are Ion exchange, Antibody-based specific purification, Use of affinity columns (Biotin or those containing specialized ligands). A summary of these solutions are: and a decision matrix on to choose appropriate options follows:

  • Choice of various isolation chemistries
  • Decision Matrix on how to choose various isolation strategies
  • Protein purification using Immunoprecipitation
  • Affinity Purification solutions

Protein purification using various methods results in diluted larger volumes of proteins that need to be concentrated and desalted to exchange buffers if more than one purification strategy needs to be implemented. Exchange of buffers is carried out by Dialysis or using Desalting columns & plates.

  • Desalting and Protein Concentration products
  • Solution for Dialysis

Choice of various isolation chemistries

Application Purity level Ligand/chemistry Base bead type Packaging options
Ion exchange purification Medium to high(application specific) Strong anion exchange
Strong cation exchange
POROS
Antibody purification High Protein A, protein G, protein A/G
Protein L
Melon Gel
Agarose, magnetic beads, POROS
Agarose, magnetic beads
Agarose
Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Fusion protein purification High Ni-NTA, cobalt, glutathione
Anti–c-Myc, anti-HA
Agarose, Superflow, magnetic beads
Agarose, magnetic beads
Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Loose resin or beads, kits
Biotin affinity purification High Avidin, streptavidin, NeutrAvidin, monomeric avidin Agarose, magnetic beads Loose resins, spin columns and kits, chromatography cartridges, 96-well spin plates
Protein immobilization High Amine reactive, sulfhydryl-reactive, carbonyl reactive, carboxyl reactive
Epoxy, tosylactivated, carboxylic acid, amine
Agarose
Magnetic bead
Loose resin or dry powder
Loose beads

Decision Matrix on how to choose various isolation strategies

Protein purification using Immunoprecipitation

Type of Product Product Name Catalog Numbers
Immunoprecipitation using magnetic beads

Antibody binding Protein A, G, A/G Secondary antibodies
(anti-mouse, anti-rabbit)
Surface-activated beads (epoxy)*
Beads only:
Dyna beads Protein A
Dyna beads Protein G
Pierce Protein A/G
Kits:
Dynabeads Protein A kit
Dynabeads Protein G kit
Pierce Protein A/G kit
Pierce Crosslink Kit (A/G)
Beads only:
Dynabeads M-280, Sheep-anti Mouse IgG
Dynabeads M-280, Sheep-anti Rabbit IgG
Kits:
Dynabeads Antibody Coupling Kit
Dynabeads Co-Immunoprecipitation Kit
Biotin binding Dynabeads™ M-280 Streptavidin
2 mL
Dynabeads™ M-270 Streptavidin
2 mL
Dynabeads™ MyOne™ Streptavidin C1
2 mL
Dynabeads™ MyOne™ Streptavidin T1
2 mL
Dynabeads™ MyOne™ Streptavidin T1
10 mL
DynaMag™-2 Magnet
1 each
11205D 65305 65001 65601 65602 12321D
Recombinant proteins Dynabeads™ His-Tag Isolation and Pulldown
2 mL
Dynabeads™ His-Tag Isolation and Pulldown
10 mL
HisPur™ Ni-NTA Magnetic Beads
2 mL
Pierce™ Anti-HA Magnetic Beads
1 mL
Pierce™ HA-Tag Magnetic IP/Co-IP Kit
40 reactions
Pierce™ Anti-c-Myc Magnetic Beads
1 mL
Pierce™ c-Myc-Tag Magnetic IP/Co-IP Kit
40 reactions
DynaMag™-2 Magnet
1 each
10103D 10104D 88831 88836 88838 88842 88844 12321D

Affinity purification solutions

Recombinant protein purification esins and magnetic beads
HisPur Ni-NTA Magnetic Beads 2 mL 88831
HisPur Ni-NTA Agarose Resin 10 mL 88221
HisPur Ni-NTA Superflow Aga ose 10 mL 25214
HisPur Cobalt Agarose Resin 10 mL 89964
HisPur Cobalt Superflow Aga ose 10 mL 25228
Pierce Glutathione Magnetic Beads 4 mL 88821
Pierce Glutathione Agarose 10 mL 16100
Pierce Glutathione Superflow Aga ose 10 mL 25236
Pierce Anti-c-Myc Agarose 2 mL 20168
Pierce Anti-HA Agarose 1 mL 26181
Biotin binding purification esins and magnetic beads
Pierce Streptavidin Magnetic Beads 1 mL 88817
High Capacity Streptavidin Agarose Resin 2 mL 20357
High Capacity NeutrAvidin Agarose Resin 5 mL 29202
Monomeric Avidin Agarose Resin 5 mL 20228
Activated support resins and magnetic beads
Pierce NHS-Activated Agarose, Dry 1 g 26196
AminoLink Plus Coupling Resin 10 mL 20501
SulfoLink Coupling Resin 10 mL 20401
CarboxyLink Coupling Resin 25 mL 20266
GlycoLink Immobilization Kit 10 columns 88941
Pierce NHS-Activated Magnetic Beads 1 mL 88826
Dynabeads M-270 Epoxy 60 mg 14301
Dynabeads M-280 Tosylactivated 2 mL 14203
Dynabeads MyOne Tosylactivated 2 mL 65501
Dynabeads M-270 Carboxylic Acid 2 mL 14305D
Dynabeads MyOne Carboxylic Acid 2 mL 65011
Dynabeads M-270 Amine 2 mL 14307D
Pierce NHS-Activated Agarose, Dry 1 g 26196
AminoLink Plus Coupling Resin 10 mL 20501
GlycoLink Immobilization Kit 10 columns 88941
SulfoLink Coupling Resin 10 mL 20401
CarboxyLink Coupling Resin 25 mL 20266

Desalting and Protein Concentration products

Type of Product Product Name Catalog Numbers
Desalting Products Zeba Spin Columns

Format Micro Spin Column 0.5 mL Spin Column 2 mL Spin Column 5 mL Spin Column 10 mL Spin Column
7K MWCO pack sizes 89877
89878
89882
89883
89889
89890
89891
89892
 89893
89894
40K MWCO pack sizes 87764
87765
87766
87767
87768
87769
87770
87771
87772
87773
Protein Concentrators Pierce protein
concentrators

Molecular Weight Cut off 3000 MWCO 5000 MWCO 10,000 MWCO 30,000 MWCO 100,000 MWCO
0.1-0.5 mL 88512 88513 88502 88503
1mL-6mL 88514
88515
88516
88517
88521
88522
88523
88524
5mL-20mL 88525
88526
88527
88528
88529
88531
88532
88533
20mL-100mL 88534 88535 88536 88537

Solution for Dialysis

Product Name Catalog Number
Complete Slide-A_Lyzer Dialysis Solutions
Slide-A-Lyzer™ G2 Dialysis Cassettes, 20K MWCO, 0.5 mL 87734
Slide-A-Lyzer™ MINI Dialysis Device, 20K MWCO, 2 mL 88405
Pierce™ 96-well Microdialysis Plate, 3.5K MWCO 88262
Slide-A-Lyzer™ Dialysis Flasks, 2K MWCO, 250 mL 87760

Protein Concentration Measurement

Most protein concentration measurements (for detection and quantitation) are carried out using methods that involve:

  • Copper chelation with the protein (Examples: BCA and Modified Lowry Protein assays) and detection of the reduce copper moieties
  • Binding of dyes to the protein and detection of protein bound dyes. Examples: Pierce 660 nm assay and Coomassie (Bradford assay).

Solutions for Protein Quantification

Product Catalog Number
Pierce™ Rapid Gold BCA Protein Assay A53225
Pierce™ BCA Protein Assay Kit 23225
Micro BCA™ Protein Assay Kit 23235
Pierce™ 660nm Protein Assay Reagent 22660
Pierce™ Coomassie Plus (Bradford) Assay Kit 23236
Pierce™ Coomassie (Bradford) Protein Assay Kit 23200
Pierce™ Detergent Compatible Bradford Assay Kit 23246
Pierce™ Modified Lowry Protein Assay Kit 23240
CBQCA Protein Quantitation Kit C6667
EZQ™ Protein Quantitation Kit R33200
Qubit 4.0 instrument Q33226
Quant-iT™ Protein Assay Kit Q33210, Q33211
Additional Links:
Total Protein Assays Overview of Protein Assays Methods

Protein standards


Protein Identification & Detection Techniques

Western analysis

  • Western analysis is a common protein identification tool. Antibodies against the protein of interest are used to detect expression of a specific protein.
  • Typically western analysis can be used to assess purity and recovery of a protein after purification steps. Alternatively, identity of a protein in a protein-protein complex can be established using this method.
  • The steps in a western are:
    • Protein separation by gel electrophoresis using a denaturing SDS-PAGE gel
    • Transfer to membranes (PVDF or nitrocellulose)
    • Detection

    Complete Western Analysis Brochure

    SDS-PAGE based protein separation

    Several forms of polyacrylamide gel electrophoresis (PAGE) exist, and each form can provide different types of information about proteins of interest. Denaturing and reducing sodium dodecyl sulfate (SDS)-PAGE with a discontinuous buffer system is the most widely used electrophoresis technique and separates proteins primarily by mass.

    By using an ionic detergent such as SDS the proteins are denatured and coated with a negative charge. Under the influence of an electric current, all the proteins migrate to the positive charged electrode based on their mass. Thus the lower mass proteins migrate faster as they are sieved through the polyacrylamide matrix and the higher mass proteins trapped in the matrix move slowly.

    Stained or unstained size markers are run on this gel to correlate size of purified products.

    Various Solutions for Gel Electrophoresis, Protein markers and Stains

    SureCast™ Gel Handcast Bundle ASureCast™ Gel Handcast Bundle A
    XCell4 SureLock™ Midi-CellXCell4 SureLock™ Midi-Cell

    Type of Product Product Name
    Gel electrophoresis bundle SureCast™ Gel Handcast Bundle A – Hardware and Reagents
    Bis-Tris Mini gels Bolt Bis-Tris Plus Gels
    NuPAge NuPage Bis-Tris Gels
    Tris-Glycine Gels Novex Wedgewell Tris-Glycine Gels
    Protein Standards iBright Protein Standards
    Gel stains Protein Stains

    The Xcell mini and midi blot modules allow gel electrophoresis of 1 to 4 gels

    Protein Transfer

    • After a protein run, the proteins in the PAGE gel are transferred via wet or semi-dye transfer to PVDF or nitrocellulose membranes
    • Wet transfer can be done manually or using an electrophoretic apparatus. Semi-dry transfer involves a special apparatus, transferring proteins in a matter of minutes. The Xcell mini and midi blot modules facilitate easy wet transfer of 1 to 4 gels. The Pierce Power Blotter can be used for a faster semi-dry transfer.
    Pierce™ Power BlotterPierce™ Power Blotter
    XCell II™ Blot ModuleXCell II™ Blot Module
    XCell SureLock™ Mini-Cell and XCell II™ Blot ModuleXCell SureLock™ Mini-Cell and XCell II™ Blot Module
    XCell4 SureLock™ Midi-CellXCell4 SureLock™ Midi-Cell

    Instruments and buffers for transfer

    BupH Tris-Glycine Transfer Buffer Packs

    SureCast™ Gel Handcast Bundle APierce Power Blotter Welcome Pack
    Product Catalog Numbers
    Pierce™ Power Blotter Welcome Pack 22834SPCL
    Pierce™ Power Blotter 22834
    Pierce™ Power Blot Cassette 22835
    Pierce™ Power Station 22838
    Pierce™ 1-Step Transfer Buffer 84731

    Western Analysis Detection

    Western Blot Detection
    Western Blot Detection

    After transfer of the proteins to the membrane, the membrane is blocked and then hybridized
    to the primary antibody which recognizes the epitope on the protein. After washing off excess primary antibody, a secondary antibody is bound, washed off. This secondary Ab contains a dye (Fluorescent, radioactive, chemiluminescent or enzyme). After washing off excess, the dye can be visualized using appropriate detection reagents & instruments.

    If the membrane needs to be re-probed to detect other protein, it can be stripped with appropriate stripping buffers and re-probed with new primary and secondary antibodies.

    40,000 different primary and secondary antibodies are available from Thermo Scientific:

    Primary Antibodies Secondary Antibodies

    Reagents for Western detection

    Blocking buffers Catalog Numbers
    Starting Block™ (PBS) Blocking Buffer 37578
    SEA BLOCK Blocking Buffer 37527
    Substrates
    1-Step™ Ultra TMB-Blotting Solution 37574
    Pierce™ ECL Western Blotting Substrate 32106
    Pierce™ ECL Plus Western Blotting Substrate 32132
    SuperSignal™ West Pico PLUS Chemiluminescent Substrate 34580
     SuperSignal™ West Dura Extended Duration Substrate 34076
    SuperSignal™ West Femto Maximum Sensitivity Substrate 34096
     
    iBright™ CL1000 Imaging System
    A32749
    Restore™ PLUS Western Blot Stripping Buffer 46430

Enzyme linked Immunosorbent assay (ELISA)

Enzyme linked Immunosorbent assay (ELISA) is an often used technique to quantitate proteins and peptides in solution. This has been used extensively in clinical applications and in basic research. 2,000 ELISA kits and reagents covering multiple species and targets are available.

A few of the cellular pathways for which solutions are provided include:

  • In-cell ELISA
  • Cytokines
  • Chemokines
  • Growth Factors
  • Neurobiological targets
  • Phosphospecific proteins and Immunoglobulins

Search for ELISA kits by Target
Reagents: Coated ELISA kits Uncoated ELISA kits
More info: Technical Resources


Protein modifications

Peptides and Proteins

Tagged and recombinant proteins and peptides are available for 9 species across various research applications. This set comprises proteins with various cellular functions.

To order and learn more visit: Other peptides and proteins

Research Category Protein Subtype
Angiogenesis
Apoptosis
Biomarker-Related Pathway(s)
Bone Research
Cardiovascular Research
Cell Cycle & Proliferation
Clinical Research
Dermatology
Development
Differentiation
DNA Replication & Repair
Drug Development
Endocrinology
Extracellular Matrix & Cell Adhesion
Gene Regulation
Genetic Diseases
Immunology
Inflammation
Metabolism
Microbiology
Neurobiology
Oncology
Other Research
Signal Transduction
Stem Cell Research
Toxicology & Drug Metabolism
Virology
Amyloid-Related Proteins
Bcl-2
BGG
BSA
Complement
DNA Binding Protein
EGF (Epidermal Growth Factor)
Ephrins
Fc Receptors
Fibronectin
Glycoproteins
GPCR Receptor
Growth Factor Receptors
GTPases
Histones
Intercellular adhesion Molecule
Lectins & Leptins
Myelin Basic Protein
Neuropeptides
Neurotrophic Factors
NFκB Pathway Proteins
Other Cytokines
Other Proteins
Phospholipases
Proteases
Proteoglycans
STAT Proteins
Tagged Protein Controls
Transferrin
Tyrosine Phosphatases
Viral Host Cell Receptors
Viral Proteins
Wnt Proteins & Receptors

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