Protein sequencing can be a very sensitive method. However, its sensitivity and usefulness are directly correlated to the quality of the sample preparation as well as the amount of sample submitted. Clearly, a sample contaminated with other proteins becomes more difficult to produce useful data as the sequence of the 'target' may be obscured by the presence of other sequences. In addition, too little material makes determination of long stretches of sequence less likely. The amount of material necessary for a successful analysis depends, in part, on the physical medium in which the sample is submitted.
Liquid Samples
Liquid samples must be at least 85% pure as determined by SDS PAGE. A minimum of 10 pmol of pure protein is required for high quality, unambiguous sequencing (depends upon the amino acids present). This amount of protein should routinely result in a clear read of 10-15 amino acids.
Samples should be free of contaminants as some chemicals interfere with the Edman Chemistry and others interfere physically in the sequencer.
Some buffer constituents to avoid are:
- non-volatile salts and buffers
- chaotropes
- glycerol
- organic solvents
- detergents
If you are unable to eliminate these materials from your buffers....PLEASE DISCUSS WITH THE PCL STAFF BEFORE YOU SUBMIT YOUR SAMPLES.
Please bring an SDS gel of your sample when sumbitting your sample.
Electroblotted Samples
Alternatively, impure samples may be separated by SDS PAGE, electroblotted to PVDF membrane and sequenced directly from PVDF membranes (nitrocellulose is unacceptable as it is not resistant to the Edman chemistry). We have recommended protocols for electroblotting and staining available on our Protocols page. The sample cartridges can hold approximately 20 square mm of PVDF membrane. This is roughly equivalent to a slice 1-2 mm high and the width of three lanes of a mini-gel. Most protein stains are acceptable (Coomassie, Ponceau S, Amido Black). We prefer that a non-glycine electroblot buffer be used. Glycine is an amino acid and will contaminate the sample resulting in uninterpretable sequence information for one or two cycles. Please contact us with any questions.
Sample Loading
Your sample will be loaded into the sequencer cartridge by spotting a pure protein liquid sample onto a Biobrene-saturated glass fiber filter or by placing a small amount of PVDF membrane directly into the sample cartridge. Liquid samples that contain contaminating or comfounding chemicals (see under Liquid Samples) will be loaded onto ProSorb cartridges and washed with 0.2% TFA to remove contaminants before sequencing commences. There is an additional charge for this preparation step.