How can I check that my protein or peptide was reduced efficiently?
There are several ways to assess the level of sample reduction. Each method expends a small amount of sample. Ellman's Reagent (Product No. 22582) allows colorimetric determination of free sulfhydryl content by comparison to cysteine (Product 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, Product No. 23030), or cyanylation (1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, CDAP), HPLC may also be used to measure sample reduction.
How do I know the Immobilized TCEP I have is still good?
The instructions that accompany the product include a protocol for testing this activity using Ellman's reagent (Product No. 22582) and free TCEP (Product No. 20490).
How long should I reduce my sample?
The incubation time required for efficient reduction in Immobilized TCEP 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. Less concentrated protein samples require shorter incubations.
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, using too little will compromise speed and/or completeness of reduction.
How quickly should I use my reduced sample?
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.
I accidentally stored the product 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.
Are other TCEP-based products available?
Yes. We offer TCEP-HCl (1 g solid, Product No. 20490) and TCEP Solution, Neutral pH (5 ml of stable 500 mM TCEP, Product No. 77720).
What factors besides time affect the rate and efficiency of reduction?
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.
What is the concentration of TCEP in the Immobilized TCEP 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 reagents decrease or interfere with the activity of Immobilized TCEP?
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.