Protein Biology

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

Additional Solutions for Protein Extraction

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

Decision Matrix on how to choose various isolation strategies

Protein purification using Immunoprecipitation

Affinity purification solutions

Desalting and Protein Concentration products

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

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

Protein standards

BSA Standard Pre-Diluted Set

BGG Protein Assay Standards

Ampule Breakers

• 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 A

  XCell4 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 Blotter

  XCell II™ Blot Module

  XCell SureLock™ Mini-Cell and XCell II™ Blot Module

  XCell4 SureLock™ Midi-Cell

  Instruments and buffers for transfer

  BupH Tris-Glycine Transfer Buffer Packs

  Pierce 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

  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

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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 are typically employed for structure elucidation and studying protein or protein-ligand studies.

Common reagents used for same are:
Crosslinking: Information & Brochure
Protein Labeling: Handbook

Cross-linker selection
Protein modification
Biotinylation
Fluorescent Reactive dyes