Monthly Archives: May 2014


Which reagent is better for reducing disulfides, DTT or TCEP? The two reagents are quite different in their reactivity, stability towards oxidation, reaction mechanisms, and other categories.


DTT is a thiol-containing reagent, and this must be considered in applications involving thiol labeling. TCEP is charged in solution and should not be used in isoelectric focusing. Aqueous solutions of TCEP are quite acidic (pH 2-3).

TCEP HCl is odorless, air-stable crystalline solid, soluble in water at a > 1 M concentrations. It reduces disulfides at room temperature in < 5 min in dilute solutions (5 -50 mM). There is no need to remove TCEP prior to the use of sulfhydryl-reactive labels or crosslinkers. TCEP is selective toward disulfides, and is reactive at a broad pH range.

For those who love Chemistry:

TCEP mechanism

Reduction of disulfide with TCEP. First step is rate-determining, kinetic rather than thermodynamic control.



Reduction of disulfide with DTT. Formation of stable cyclic disulfide drives the reaction.

You can learn more about DTT and TCEP from this article: “A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry”, Elise Burmeister Getz et al.  Analytical Biochemistry 273, p. 73–80 (1999)

2D PAGE in less than one page

Approximate timeline

Day 1

Immobilized pH gradient (IPG) strip rehydration: active time 5 min per sample, rehydration is carried out overnight

Day 2

Isoelectric focusing (IEF): active time 5 min per sample, IEF 5-6 hrs.

Preparation for the 2nd dimension SDS PAGE: 1 hr.; necessary reagents and solutions are prepared during the last hour of IEF.

SDS PAGE, staining, and de-staining: 2.5 hrs. with de-staining overnight.


Brief procedure (learn more in the 2D PAGE manual )

Day 1 (late afternoon)

Protein sample should be lyophilized or precipitated. To keep the ionic strength of the protein solution at minimum, avoid salts. Non-ionic and zwitterionic solubilizing agents could be present, but keep in mind solubility limits.

Protein load for a complex sample to be stained with Coomassie is approximately 100 ug. The protein load varies depending on many factors including stain sensitivity, IPG range, or downstream applications (refer to the manual).

Protein sample is dissolved in the sample buffer (Bio-Rad cat #163-2106), 125 ul for 7 cm IPG strips and 185 uL for 11 cm IPG strips. The protein solution is pipetted into a channel in a rehydration tray. An IPG strip is placed gel side down into the channel, covered with mineral oil, and left overnight at 4 C.


Day 2 (early morning)

IPG strips are placed into a focusing tray, covered with mineral oil, and the tray is placed into the IEF cell for 5-6 hrs.

After the IEF is complete, strips are soaked in a reducing buffer, followed by alkylating buffer, rinsed in SDS running buffer, and placed at the top of an SDS-PAGE gel. A molten agarose solution is applied to the well. Once agarose solidifies, the gel is ready for the 2nd dimension electrophoresis.

After completion of the SDS-PAGE, gels are rinsed, stained, and destained. Alternatively, the gels can be electro-blotted.


Immobilized pH gradient (IPG) strips

What IPG strip is the best for your experiment? Start by choosing the length (7 cm or 11 cm). Short strips are compatible with the Mini-PROTEAN cell, longer strips are compatible with the Criterion cell. All parameters being equal, a larger gel affords better resolution. Next select pH range depending on a type of your experiment: broad range (e.g. 3-10) for a global view, narrow range for a zoom-in view.
Bio-Rad cat #163-2000, 7 cm, pH 3–10, immobilized pH gradient (IPG) strip for first-dimension separations, pkg of 12
Bio-Rad cat #163-2014, 11 cm, pH 3–10, immobilized pH gradient (IPG) strip for first-dimension separations, pkg of 12

Buffers and gels

Rehydration/sample buffer, Bio-Rad cat #163-2106

Agarose, Bio-Rad cat #163-2111

Criterion Tris-HCl Gel, Bio-Rad cat #345-0040, Pkg of 1, 8–16% polyacrylamide gel, prep+2 well, 800 μl, 13.3 x 8.7 cm (W x L), for use with Criterion and Criterion Dodeca cells

10x Tris/Glycine/SDS, Bio-Rad cat #161-0732, 1 L, 10x premixed electrophoresis buffer, contains 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 following dilution to 1x with water

SDS equilibration buffer II: 6M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, Bio-Rad cat #163-2108

And for the best results: Electrode Wicks, Bio-Rad cat #165-4071, Pkg of 500, precut electrode wicks, for use with the PROTEAN® i12™ IEF system. (I still have about 450, no need to buy)

2D PAGE video tutorial

2D PAGE detailed procedure for the Bio-Rad starter kit

Please let me know if you are interested in a 2D PAGE experiment!


May 27, 2014

Starting tomorrow, May 28th and through June 4th we will be hosting a mass spectrometry practicum as a part of the Penn State Bioinorganic Workshop. Orbi will be used by our colleagues from Northeastern University to demonstrate the power of high-resolution MS in the analysis of metalloenzymes. I will not be able to run samples on Orbitrap during the workshop.

Open-access LCMS

The Mass Spec Facility must regretfully announce that the open access liquid chromatograph/mass spectrometer is no longer operational.  The instrument, an AB Sciex 150EX single quad mass spectrometer, was purchased in 2006, and has been nearing the end of its life span.  The instrument is no longer supported by the manufacturer, so parts and qualified service will be increasingly hard to find.  Thus, it is not possible for the facility to maintain.

Nominal mass samples can still be submitted to the facility and will be given high priority for quick turnaround (generally 24 hrs. or less).  Prior to the purchase of the 150EX the facility routinely ran nominal mass samples in this manner.  Contact James Miller ( with any questions you might have.

Thank you.

Plate reader

The SpectraMax M5 plate reader which used to be in the Millennium Science Complex, W-124A is now located in the Proteomics and MS Core Facility, in 3 Althouse. Please contact Tatiana to reserve the plate reader time:, 814-863-3503. There is currently no charge for using this plate reader.

The SpectraMax M5 Multi-Mode Microplate Reader delivers single mode reader performance in one multimode reader package. The dual monochromator optics allow the widest range of applications to be utilized for bioresearch and drug discovery applications, all without the need to change filters. The SpectraMax M5 Multi-Mode Microplate Reader possesses the five modes preferred by the majority of our users providing the benefit of multiple detection modes in one platform:

UV-Visible Absorbance (Abs)

Fluorescence Intensity (FI)

Time-Resolved Fluorescence (TRF)

Fluorescence Polarization (FP)

Glow Luminescence (Lumi)

User Manual – SpectraMax M3 M4 M5 M5e

Software Manual – SoftMax Pro

New use rates

As the number of Facility customers grows, the turnaround times are getting longer. The two major factors that slow us down are sample preparation and data interpretation.

The time spent on preparing and cleaning your samples is the time taken away from MS-related activities, e.g. operating and maintaining the equipment, processing and interpreting data, and developing better methods for your samples. Sample prep is something we can delegate to you, and many of our customers have been submitting MS-ready samples and getting their results quickly.

The new use rates are designed to save you money and time should you decide to prepare your own samples. This does not mean that we will no longer prepare your samples; it simply means that you will be billed for the staff time in addition to the instrument time if we have to spend a significant amount of time working on your samples.

To help you overcome beginner’s fright, I can present a series of 1-hr hands-on sample preparation workshops, which will be offered as needed (i.e. as soon as 5 people express interest in the same workshop topic). The cost will be $57 per person, and the protocols will be freely available on-line before the workshop announcement so that you can decide whether you wish to attend. I am open to suggestions for the first workshop, e-mail me at with the subject line ‘workshop topic’.


May 6, 2014

Orbi is running Juan’s, Qusai’s, and Shaima’s samples. Joana’s first three samples will go next. A lot of housekeeping work in preparation for the new instrument, but we are getting there.


May 5, 2014

Are we there yet? Are we there yet? Well, let’s see where we are…

MALDI-TOF-TOF is scheduled to arrive next week, and there is a list of things that need to be done so that the instrument installation starts without a delay.

I have 14 samples on my bench, all of which I am preparing for my customers. I also have 6 samples in the queue, which will run as soon as I calibrate the instrument.

If your experiments require short turnaround times, submit samples that are ready for the MS analysis, otherwise – please wait for your turn in the queue!

In-solution digestion of proteins

Purified proteins or protein mixtures can be digested in solution if an additional separation step is undesirable or unnecessary.

Proteins in solution are usually denatured by boiling or using denaturing buffers. During this step, the disulfide bonds must be reduced, and the sulfhydryl groups must be alkylated to prevent the disulfides from re-forming. The protein samples are then incubated with trypsin for several hours, and the resulting peptides can be analyzed by MS.

Denaturing buffers contain chaotropic agents, salts, and detergents at concentrations that inactivate trypsin. Before adding trypsin, you should desalt your protein sample and remove detergents. There are a number of the detergent removal and desalting options: detergent removal spin columns, size-exclusion and MW-cut-off spin columns, ion-exchange membranes and resins, etc.  Gel-assisted proteolysis is another option, but then it is not really an ‘in-solution’ procedure, although it does not involve electrophoresis. I thought I should mention it here in case it could be of interest to you.

You will need

Digestion buffer: 16 mg/mL ammonium bicarbonate in water

Reducing reagent: 30 mg/mL TCEP (~100 mM, Sigma C4706)  or 15 mg/mL DTT (Sigma D0632 ) in digestion buffer. NOTE: The 30 mg/mL TCEP stock solution must be prepared in 16 mg/mL (~200 mM) ammonium bicarbonate to bring up its pH. The final concentration of TCEP in the digestion mixture should be 5-10 mM.

Alkylating reagent: 18 mg/mL iodoacetamide (Sigma I1149) prepared fresh in the digestion buffer

Proteomics grade trypsin (e.g. Sigma T6567-5x20UG or Thermo Pierce 90057, 5 vials x 20 ug lyophilized powder). Trypsin, 20 ug can be dissolved in 20 uL of 1 mM HCl or 50 mM acetic acid, pH ~ 3, aliquoted and stored at -20C (stock solution).

To prepare activated (or working) trypsin solution, dilute trypsin stock solution with digestion buffer 10-fold to 0.1 ug/uL concentration.


Volumes are approximate, it is a sample procedure after all. Trypsin should not exceed 5% of the total protein, provided the protein concentration range is known.

Combine 15 uL digestion buffer, 3 uL reducing reagent, and up to 12 uL sample solution containing 0.025 – 10 ug protein (total volume 30 uL)

Denature/reduce at 50-60 C (TCEP) or in a boiling water bath (DTT) for 5 – 10 min, cool to r.t., spin down to collect the sample

Add 3 uL alkylating reagent and incubate in the dark at r.t. for 20 min

If protein sample contains detergents, salts, or chaotropic agents, perform buffer exchange after the alkylation using a 3,000 MWCO centrifugal filter. It will be impossible to remove detergents after the digestion; and most detergents are not compatible with LC MS analysis. You can find a list of MS-compatible detergents here.

Add 1-5 uL activated trypsin and incubate at 37 C for 3 hrs. Optional: add 1-5 uL of fresh activated trypsin and incubate for an additional 2 hrs at 37 C or overnight.

Once the incubation is complete, the peptides can be submitted for analysis or stored at -20 C.

Helpful tips

Minimum sample amount required for MS analysis is in the fmol/uL range (ng/uL). Solutions of peptides at very low concentrations (e.g. less than 100 fmol/uL) should not be stored for more than 1-2 days.

Always run a control along with your sample. It could be a 1 mg/mL solution of bovine serum albumin or other standard protein that you have in your lab prepared in the same buffer as your sample and taken through the entire procedure. We don’t charge for analyzing your controls.

TCEP is a great reducing reagent because it does not contain -SH groups and thus does not consume iodoacetamide during alkylation, unlike DTT. TCEP solutions in water are acidic. Depending on your buffer composition, you might observe your sample coming out of the sample tube as a soapy foam the moment you add TCEP. It is pretty much impossible to put that foam back into the tube, don’t ask me how I know. So, prepare your TCEP solutions in ammonium bicarbonate buffer to get a pH close to 8. Another note about TCEP: it should never be stored in phosphate buffers because it quickly decomposes in the presence of phosphate.

Have I missed anything? Let me know!