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Thermo Scientific™ Pierce™ Immobilized TCEP Disulfide Reducing Gel

Catalog No. PI77712
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PI77712 5 mL
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Catalog No. PI77712 Supplier Thermo Scientific™ Supplier No. 77712
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Description

Efficiently reduce protein or peptide disulfide bonds with this stable, odorless, beaded agarose resin on which Tris (2-carboxyethyl) phosphine is immobilized.

Thermo Scientific™ Pierce Immobilized TCEP Disulfide Reducing Gel is Tris(2-carboxyethyl)phosphine that is covalently immobilized to beaded agarose resin to enable efficient reduction of peptide and protein disulfide bonds.

Trialkylphosphines serve as excellent agents for the reduction of disulfide bonds in proteins, peptides and other disulfide bond-containing molecules and are relatively unreactive toward other functional groups. The trialkylphosphine TCEP is stable in aqueous solutions and does not undergo the rapid oxidation that often occurs with other reducing agents such as dithiotreitol (DTT) and β-mercaptoethanol (BME). TCEP does not interfere with common sulfhydryl-reactive reagents (e.g., maleimide crosslinkers). Nevertheless, many protocols require recovery of the reduced sample separate from the reducing agent. Immobilized TCEP Disulfide Reducing Gel eliminates the need to use laborious and inefficient gel filtration methods to separate the reduced sample from the reducing agent. Immobilized TCEP Disulfide Reducing Gel may be adapted conveniently to a variety of scales and formats (i.e. batch, spin column and gravity-flow column procedures).

Highlights:

  • Odorless: reduce proteins at your desktop; contributes to a healthier lab environment
  • Stable: virtually no loss of activity when stored refrigerated
  • Versatile: reduces peptides and proteins over a broad range of pH, salt, detergent and temperature conditions
  • Flexible: resin slurry provides flexibility for use in spin tube, column or batch modes
  • Time-saving: no soluble reductant removal saves time and conserves sample
  • Efficient: reverses both natural disulfide bonds and disulfide bonds artificially incorporated by crosslinking; excellent alternative for uncoupling crosslinked proteins or peptides by agents such as DSP and DTSSP

Specifications

Content And Storage Upon receipt store at 4°C. Do not freeze.
Product Line Pierce
Form Liquid
Quantity 5 mL
Product Type Disulfide Reducing Reagent

Frequently Asked Questions (FAQs)

What reagents decrease or interfere with the activity of Immobilized TCEP Disulfide Reducing Gel?

TCEP is more tolerant of nickel and cobalt than other reducing agents, such as DTT. However, TCEP is inactivated by other metals, namely copper, magnesium, silver and zinc. For this reason, avoid using metal utensils and instruments when working with Immobilized TCEP. Adding EDTA (5-20 mM) to the sample buffer will help maintain activity of the Immobilized TCEP by chelating many divalent metals. In addition, cyanate compounds will react with free sulfhydryls, making them unavailable for intended applications. Because urea can form cyanate degradation products, its use in the sample is not recommended.
What is the concentration of TCEP in the Immobilized TCEP Disulfide Reducing Gel?

The concentration of TCEP is approximately 8 mM in the settled resin. However, take care when attempting to use this value to calculate the gel amount necessary for sample reduction because the reducing capacity of Immobilized TCEP is somewhat less than an equal concentration of free (non-immobilized) TCEP. Steric hindrance and diffusion to the immobilized TCEP affect the efficiency of interaction between sample molecules and the reductant.
What factors besides time affect the rate and efficiency of reduction using the Immobilized TCEP Disulfide Reducing Gel?

Increasing the temperature will help to accelerate reduction, e.g., 30 minutes at 37°C is roughly equivalent to 1 hour at room temperature. For proteins, the efficiency of reduction may be improved by changing to more acidic or alkaline conditions (pH 3-4 or pH 8-9), which can alter protein conformation sufficiently to render certain disulfides more accessible to the reducing agent. Likewise, the addition of a denaturant (e.g., 3 M guanidine-HCl) or detergent can expose otherwise inaccessible disulfides to the immobilized reducing agent.
Are other TCEP-based products available besides Immobilized TCEP Disulfide Reducing Gel?

Yes. We offer TCEP-HCl (1g solid, Cat. No. 20490, 10g solid, Cat. No. 20491), TCEP-HCl 10 x 1 mg No-weigh format, Cat. No. A35349 and TCEP Solution, Neutral pH (5 mL of stable 500 mM TCEP, Cat. No. 77720).
I accidentally stored the Immobilized TCEP Disulfide Reducing Gel at room temperature rather than 4°C. Is it still good?

TCEP is fairly stable, even at room temperature. If stored in the original container free of contamination, the product likely retains its full activity. If uncertain, test the activity by the protocol mentioned above and described in the instructions for the Immobilized TCEP Disulfide Reducing Gel.
How quickly should I use my sample reduced with the Immobilized TCEP Disulfide Reducing Gel?

Disulfide bridges can reform quickly in samples that have been reduced but are no longer in the presence of the reducing agent. The tendency of -SH groups to reform disulfides after reduction is dependent on the concentration of free -SH groups generated and the elapsed time after reduction. Therefore, procedures utilizing the reduced sample should be performed immediately after reduction. Including 5-20 mM EDTA in the sample buffer during reduction will help prevent oxidation of the generated sulfhydryl groups.
How much Immobilized TCEP Disulfide Reducing Gel should I use?

In general, a volume of gel equal to 0.5-1.0 X the sample volume is sufficient to ensure timely and efficient sample reduction. For example, use 50-100 mL of packed Immobilized TCEP Gel for 100 mL of sample. Although it may be possible to use less Immobilized TCEP Gel, using too little will compromise speed and/or completeness of reduction.
How long should I reduce my sample using the Immobilized TCEP Disulfide Reducing Gel?

The incubation time required for efficient reduction in Immobilized TCEP Disulfide Reducing Gel depends on the type of sample. Peptides will be completely reduced at room temperature in 10-15 minutes (i.e., about the amount of time for a sample to pass through a gravity-flow column). Proteins may contain disulfides bridges that are less accessible and will require longer incubation times to achieve thorough reduction. Even so, incubation for 30-60 minute is usually sufficient. Incubation for longer than 2 hours is not recommended because the rate of disulfide formation will begin to exceed the rate of their reduction, resulting in a net decline in reduction compared to the same sample incubated for a shorter time. Protein concentration will also affect the rate of reduction. Samples at concentrations greater than 1 mg/mL will usually require 1 hour incubation with the Immobilized TCEP Disulfide Reducing Gel. Less concentrated protein samples require shorter incubations.
How do I know that the Immobilized TCEP Disulfide Reducing Gel I have is still good?

The instructions that accompany the product include a protocol for testing its activity using Ellman's reagent (Cat. No. 22582) and free TCEP (Cat. No. 20490).
How can I check that my protein or peptide was reduced efficiently using the Immobilized TCEP Disulfide Reducing Gel?

There are several ways to assess the level of sample reduction. Each method expends a small amount of sample. Ellman's Reagent (Cat. No. 22582) allows colorimetric determination of free sulfhydryl content by comparison to cysteine (Cat. No. 44889) standards. If appropriate control lanes are included in the gel, sample reduction can be assessed by SDS-PAGE using non-reducing conditions. After capping the free sulfhydryls by alkylation, N-ethylmaleimide (NEM, Cat. No. 23030), or cyanylation (1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, CDAP), HPLC may also be used to measure sample reduction.
Can other reducing agents other than DTT or BME be used to reduce proteins prior to electrophoresis? For example, what about TCEP (Tris Carboxy Ethyl Phosphene)?

TCEP, Tris Carboxy Ethyl Phosphene is an alternative sulfhydryl reducing agent for protein samples. It is an extremely potent and effective reducing agent for particularly ‘difficult' proteins. It is compatible with the Tris-Glycine gels and NuPAGE gels. It should be added to the sample buffer for these systems. 20 mM final (maximum) concentration is sufficient for samples. You may add alkylating agents, e.g. Iodine (50 mM Iodoacetic acid), to prevent re-forming of S-S bonds but it is not necessary. Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.

For Research Use Only. Not for use in diagnostic procedures.