Monthly Archives: December 2013

Extracting large peptides from gel after digestion

First, let’s define ‘large’. The Proteome Discoverer sets the upper limit for a precursor ion at 10,000 Da. This means anything bigger than 10 kDa will not be considered even if it’s present in the MS data, and a different software package ($$) will be required to analyze the high MW data. Clearly, proteolytic peptides with MW > 10 kDa will not be very useful for protein identification. I suggest using a different enzyme or a combination of enzymes. I have seen tryptic peptides up to 7 kDa in some in-gel digested samples, so apparently some large peptides do come out of gel.

Next, a large peptide’s physico-chemical properties (e.g. hydrophobicity, pI, hydrodynamic radius) must be considered as they will affect the extraction efficiency. If the peptide’s properties are known, the extraction solvent composition and pH can be adjusted to improve the peptide’s solubility.

Finally, let’s consider the gel from which the large peptides need to come out. Obviously, it will be easier to get the large peptides out of a 4 %T gel than out of a 20 %T one. Soaking a gel piece in deionized water and then freezing it should crash enough pores in the gel to improve the extraction  of large peptides (water expands as it freezes). Additionally, the gel could be ‘squeezed out’ a few times by changing extraction solvent from neat acetonitrile to an aqueous mixture. The gel piece will shrink in acetonitrile expelling the peptide solution. Re-hydrating the gel and then shrinking it again in acetonitrile will ‘squeeze out’ more of the digest.

Using elevated temperature (50 C), vortex mixer, and/or ultrasonic bath should all improve the extraction. Use common sense: 50C and a high pH buffer is not a good idea for the phosphopeptide extraction. Another word of caution: don’t get carried away. Three extraction steps should be enough. If you end up with a large volume (e.g. more than 0.5 mL), the benefits of a thorough extraction might become negated by the losses due to dilution. Peptides and proteins tend to adhere to the polypropylene tubes. A large volume of a dilute peptide solution presents a large surface area for the peptides to adsorb.

What to do if this doesn’t work? You can try in-solution digestion. If the mixture is too complex and a PAGE step is necessary, you can try electroeluting the protein(s). Intact proteins electroeluted from gel bands can be buffer-exchanged using small-volume 3,000 Da MWCO spin columns and proteolyzed in solution.


December 13, 2013

Tyler, who is not afraid to submit his samples on Friday, the 13th, should get his Orbi data by Monday next week if all is well in our haunted building. Have a nice weekend!


December 11, 2013

Orbi is running samples for Eric tonight. Eric is a student at Hershey.

M@LDI LR (aka Matilda) has left the facility today to join other Matildas of her age.

The Winter Holiday schedule is up on our door: we will be closed from Monday, December 23rd, through Friday, January 3rd, and back to work on January 6th. Please plan accordingly!

Gel bands and gel bandits

A protein ID confirmation is probably the most requested proteomics service in the facility; and it is not just a good (or ‘expensive’) idea – it could save you hours in the lab in the long run. When the MS result is not what you expected, don’t panic: use the MS information to your advantage! Knowing the parameters of interfering proteins (MW, pI, mechanism of metal ion binding) can help you to optimize or change your purification scheme.  

…Poly-His purification approach was inspired by high affinity of transition metal ions (divalent Co, Ni, Zn, and Cu) for His and Cys residues in naturally occurring proteins way back in 1975 …

What I usually see is a prominent gel band that is thought to contain a protein of interest with a poly-His purification tag which has been expressed in E.Coli and purified on an immobilized metal affinity chromatography (IMAC) column. When the protein of interest ID is confirmed, you leave me with a smile and a thank you, so read no further.

This post is for my disappointed customer whose gel band got hijacked by a bunch of E. Coli gel bandits. Having seen enough of their sneering mug shots/accession numbers, I have compiled a quick reference list of these interfering so-and-so’s from the references 1 and 2 (also included as pdf). Please note that these accession numbers are for K12, other strains will have a different accession number for the same gene product. Additional references, some of which offer solutions to the interference problem(s), are included for your enjoyment. If you encounter recurring interfering proteins in your purification system, and they are not listed here, please share this information!

Gel bandits reference list



  1. Bolanos-Garcia and Davies; Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. doi:10.1016/j.bbagen.2006.03.027
  2. Bartlow et al.; Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level. doi:10.1016/j.pep.2011.04.021
  3. Robichon et al.;Engineering Escherichia coli BL21(DE3) derivative strains to minimize E.coli protein contamination after purification by immobilized metal affinity chromatography. doi:10.1128/AEM.00119-11
  4. Parsy et al.; Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography. doi:10.1016/j.jchromb.2007.03.046
  5. Block et al.; Immobilized-Metal Affinity Chromatography (IMAC): A Review. Methods in Enzymology, doi:10.1016/S0076-6879(09)63027-5

Gel bandits reference list pdf


Highlights from today’s seminar

Direct analysis of TLC spots by MALDI-TOF

Slide1 Slide2 Slide3


“Edmass” sequencing, N-terminal and C-terminal sequencing of intact SOD1 protein

Slide1 Slide2 Slide3


RNA oligomer (28-mer) MW and sequence analysis – on just 1 microliter of sample

Slide1 Slide2 Slide3 Slide4


PolyTools software screenshot



Thank you all for participating and asking questions!