PROTEIN SEQUENCING
N terminal Protein sequencing by Edman Degration and protein manipulation services (such as digesting a gel slice and obtaining a sequence) can be provided. Protein sequencing services can be provided with a usual turnaround time of 1 week.



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Sample Requirements

The required mass of the peptide/protein and the formats allowable depend on several variables, and these variables will determine the ultimate outcomes.  An ideal peptide (one that is purified and which does not wash off the cartridge matrix) can be sequenced for 20 to 25 cycles at low pmol levels (10 to 15 pmol). However, such samples are scarce. In fact, buffers, detergents, and other peptides present, as well as the sequence itself, will affect the results.  Copious amounts of Tris and Glycine, salts, as well as detergents, will also adversely affect the ability to interpret the data or interfere with machine operation.
For PVDF blotted samples, the “rule of thumb” is that a well defined, clearly visible coomassie stained band blotted to PVDF will generally yield some information.  For solution samples, 50 pmol or so is recommended for strong results; however, samples with 10 to 20 pmol will produce adequate results.  
While these recommendations clearly do not reflect current state-of-the-art analysis, the PPF finds that “real-world sequencing” is subject to unpredictable variables; therefore, estimates of producing robust results must be made on the conservative side.  Naturally, it is assumed that the NH2-terminus is not blocked.


Submitting Your Samples

Bring your sample to the Purdue Proteomics Facility located in Room B054C in the Hansen Building.  Fill out a Protein Sequence Analysis Request Form, which can be found on the wall outside the lab.  Leave the form and your sample inside the facility with one of the lab technicians.

OR

Download the Protein Sequence Analysis Request Form here and return it to the Purdue Proteomics Facility with your sample(s) to be sequenced.
The Protein Sequence Analysis Request Form will require a valid account number, your name, your telephone number, and information about the sample, including how far you want it sequenced (note: you will get one amino acid per cycle).  If you are just checking to see if you have isolated the desired known, indicate just 5 cycles.  If this is a precious and unknown sample, just say "All Possible,” and as much readable sequence as possible will be attempted.
Turnaround times vary due to demand, but it is generally one week.


Materials and Equipment

The Purdue Protein Facility uses the Applied Biosystems Procise 492 Dual Column instrument. This is a completely integrated, computer-controlled device capable of several modes of operation with either blotted or solution (glass fiber matrix) samples.  Both gas-phase and pulsed-liquid phase cycles are operational.  Data collection is digital, and files can be downloaded to a laser printer for image/data manipulation.  There are extra bottle positions for experimental chemistries.


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