Shop All Denaturing Agents

Guanidine Isothiocyanate (Invitrogen™)

UltraPure™ Guanidine Isothiocyanate is a strong protein denaturant when used at high concentrations. It is useful in the isolation of intact RNA from sources rich in RNase such as pancreas (1).

Inclusion Body Solubilization Reagent (Thermo Scientific™)

The Thermo Scientific Inclusion Body Solubilization Reagent effectively retrieves recombinant proteins that form insoluble aggregates during bacterial overexpression.

Features of Inclusion Body Solubilization Reagent :

• Achieves the essential first step for subsequent insoluble protein refolding procedures
• One easy- and ready-to-use solution; no preparation needed
• Compatible with Thermo Scientific Pierce Coomassie Plus Protein Assay Kit (Part No. 23236) to determine degree of expression
• Proprietary denaturant can be easily removed by dialysis so that SDS-PAGE can be performed
• Can accommodate additional components such as reducing agents or chelating agents

The Inclusion Body Solubilization Reagent is a ready-to-use aqueous solution of denaturant that helps to dissolve aggregated proteins (inclusion bodies) that have been separated from the soluble fraction of bacterial cell lysates. The reagent is designed to retrieve expressed protein in soluble form after lysis and extraction with reagents such as Thermo Scientific B-PER Bacterial Protein Extraction Reagent. Bacteria are widely used for recombinant protein expression. However, 70%-80% of proteins produced by recombinant techniques in E. coli form inclusion bodies (i.e., protein aggregates). Once these aggregates are formed, it is very difficult to solubilize them. A proprietary denaturant contained in this reagent provides the most effective means for solubilizing aggregated proteins. Additional components, such as a reducing agent and a chelating agent, may be added to the reagent, depending on the particular application.

UltraPure™ Sodium Dodecyl Sulfate (SDS) (Invitrogen™)

UltraPure™ Sodium Dodecyl Sulfate (SDS) is a detergent that functions as a protein denaturant. It is used in the determination of protein molecular weight by polyacrylamide gel electrophoresis. It is also widely used to disrupt cell walls and dissociate nucleic acid-protein complexes in DNA and RNA extraction protocols.

ZOOM™ Urea (Invitrogen™)

The ZOOM® IEF Fractionator offers a fast, reliable method to reduce sample complexity, enrich low abundance proteins, and increase the dynamic range of detection. Solution phase isoelectric focusing with the ZOOM® IEF Fractionator provides reproducible separations in three hours. Fractionated samples are ready for further analysis by two dimensional gel electrophoresis (2DE), one dimensional gel electrophoresis (1DE), or two dimensional liquid chromatography/mass spectrometry (2D LC/MS).

The ZOOM® IEF Fractionator System (Figure 1) consists of the following components:

• ZOOM® IEF Fractionator is built for safety, reliability, and durability. It includes easy-to-assemble sample chambers, in-built buffer chambers, buffer trough, removable electrode assembly, and safety lid.
• ZOOM® Disks are pre-cast polyacrylamide gels that eliminate the need for manual preparation and minimize any chance of cross contamination. These immobilized buffered disks are pre-labeled, disposable, and designed for single use, ensuring consistent and reproducible fractionation. They resolve samples into as many as 7 fractions (using 8 disks of specific pH) to six fractions (using six disks of specific pH) from pH 3-12. Simply place each disk between the sample chambers in the ZOOM® IEF Fractionator to allow separation in a specific pH range.
• ZOOM® Reagents are proteomic grade reagents, including ZOOM® 2D Protein Solubilizers urea, thiourea, CHAPS, and ampholytes, that ensure optimal conditions for sample preparation.
• Basic Protein Kit which includes ZOOM® Discs and ZOOM Strips for IEF of basic proteins, focusing buffers, and solubilizer kits.

Sample Chamber and Fractionator Unit Set-up Procedure
Figures 4-6 illustrate assembly of the sample chambers and fractionator unit. The sample chamber and components fit together easily and offer a leak-proof seal. Simply place disks between the sample chambers in the ZOOM® IEF Fractionator to allow separation in a specific pH range. For example, if you want to fractionate proteins between pH 4.6 and 5.4, flank a sample chamber with a ZOOM (r) Disk of pH 4.6 and one of pH 5.4.

Downstream Processing by 2D Gel Electrophoresis
Following fractionation, the separated samples can be subjected to further analysis using narrow or broad range ZOOM® Strips on the ZOOM® IPGRunner™ System. Follow with second dimension analysis using neutral pH NuPAGE® ZOOM® Gels.

Pierce™ Protein Refolding Kit (Thermo Scientific™)

The Thermo Scientific Pierce Protein Refolding Kit includes high-purity reagents and detailed instructions for using a matrix strategy to determine optimal buffer conditions for refolding recombinant proteins that have been denatured and solubilized from inclusion bodies.

Features of the Protein Refolding Kit:

Robust—conditions and components examined are limited to those having the most significant and general utility as folding buffers
Convenient—three-level matrix design significantly reduces the amount of secondary optimization required and increases the ease of data interpretation
Adjustable matrix format—allows refolding experiments to be customized to the target protein; known positive and negative interactions between buffer components are addressed, minimizing unnecessary analyses
High-purity reagents—reagents are formulated using stringent standards so that consistent results are attained

The kit contains the essential reagents and complete strategy for determining optimal buffer conditions to refold denatured recombinant proteins to restore native structure and function. Nine base refolding buffers form a matrix that includes a range of strong and weak denaturant conditions for the suppression of protein aggregation. The supplied additives are used as additional matrix factors, depending on the protein type being refolded. Buffer components are examined at three concentration levels, allowing a wide spectrum of folding conditions to be tested within one experiment. The adjustable design allows matrix conditions to be tailored to the target protein, preventing sample waste and unnecessary analysis, while maximizing refolding yields. The Pierce Protein Refolding Kit is accompanied by a comprehensive Refolding Guide with details on isolating, solubilizing and purifying inclusion bodies; optimizing refolding conditions; and analyzing refolding yields.

Note: The following section is based on the original Previews article that reported on the development of the Pierce Protein Refolding Kit, which was originally called "Pro-Matrix".

Lysozyme was denatured overnight at 4°C in 8M GdnHCl, 10 mM DTT, 50 mM Tris, pH 8.0 at 20 mg/mL. Reduced glutathione, oxidized glutathione and DTT were added to refolding buffers as determined by the matrix layout (Table 3). All solutions were equilibrated to 4°C. Immediately before adding solubilized lysozyme to refolding buffers, the DTT was removed using a protein desalting spin column equilibrated in 8M GdnHCl, 50 mM Tris; pH 8.0. Lysozyme was then added to the refolding buffers at a final concentration of 1 mg/mL. This addition supplies 0.4M GdnHCl to the Base Refolding Buffers. Refolding was allowed to proceed for 18 hours at 4°C. Refolding yields were determined by measuring lysozyme activity with the EnzChek(tm) Lysozyme Assay Kit (Molecular Probes) using a Tecan(tm) SPECTRAFluor Plus System.

Results and Discussion
The basic protocol for protein refolding requires that inclusion bodies are first isolated, purified and then solubilized with a strong denaturant, such as guanidine hydrochloride (GdnHCl), to produce a completely unfolded protein. The solubilized protein is then diluted or dialyzed into a refolding buffer to reduce the denaturant concentration, allowing the protein to refold based on the information contained in its primary sequence. When using optimized conditions many proteins can be reliably refolded at concentrations >1 mg/ml. However, if the denaturant is removed and replaced with a non-optimized refolding buffer, protein aggregation strongly competes with renaturation and only minimal amounts of native protein are recovered. The degree of aggregation that occurs during refolding is largely dependent on protein concentration, concentration of strong and weak denaturants, pH, temperature, and the redox environment. Ionic strength, divalent cations, polymers and cofactors can also promote refolding of some proteins.

The results for refolding reduced and denatured lysozyme using the Pierce Protein Refolding Kit are reported in Table 3. For this example we treated lysozyme as if the presence of disulfide bonds in the native state was unknown. Reformation of native lysozyme was suppressed at the lowest and highest denaturant concentrations present within the protein refolding matrix (trial numbers 1, 2 and 9). Refolding was also suppressed by the presence of DTT (trial numbers 1, 6 and 8), showing the importance of reforming disulfide bonds in the folding of lysozyme. Highest lysozyme activity regained in the experiment was achieved in trial seven, which contained 1.4M GdnHCl, 0M L-arginine, 2 mM GSH: 0.4 mM GSSG, and represents more than 90% of the solubilized lysozyme being refolded.

UltraPure™ Guanidine Isothiocyanate Solution (Invitrogen™)

UltraPure™ Guanidine Isothiocyanate Solution consists of 4 M guanidine isothiocyanate, 50 mM Tris-HCl (pH 7.5), 25 mM EDTA. It may be used as part of a protocol, based on the guanidine isothiocyanate procedure first described by Chirgwin et al. (1) and later modified by Han et al. (2), for isolating intact mRNA from tissues or cultured cells.

8M Guanidine-HCl Solution (Thermo Scientific™)

Thermo Scientific Pierce 8M Guanidine-HCl Solution is an accurately formulated, ready-to-use solution of guanidine hydrochloride, that can be easily diluted and pH-adjusted to any concentration below 8M.

Features of 8M Guanidine-HCl Solution:

• Any concentration below 8M guanidine can be quickly and easily prepared.
• No wasting valuable research time weighing and dissolving guanidine crystals
• Free from UV absorbing materials in the range of 225-300nm
• Free from heavy metal contaminants
• Free from degradation products such as ammonia
• Excellent stability

Applications of Guanidine Hydrochloride:
• Solubilizing proteins from inclusion bodies
• Increasing solubility of hydrophobic peptides and proteins
• Denaturing proteins

Chaotropes, substances that disrupt the structure of water interactions, help to solubilize hydrophobic proteins and peptides for a variety of biological purposes. For example, hydrophobic peptides and proteins may be dissolved in coupling buffer containing either 6M guanidine-HCl or 4M urea prior to immobilization on AminoLink Plus Support to create an affinity column. However, because the degradation products of guanidine and urea include ammonia, which contains a primary amine that will compete for reaction to the support, urea and guanidine solutions must be prepared with high quality reagents immediately before use.

Guanidine is a general protein denaturant, unfolding proteins and altering their three-dimensional structure. Consequently, some proteins will be irreversibly altered upon interaction with guanidine solutions and may lose their binding function. Before any large-scale use of guanidine, it is best to test a small sample and determine whether the denaturing effects will adversely affect the intended use of the protein.

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