Sample Requirements

Sample Requirements: MICROSEQUENCING ON PVDF MEMBRANES

Dear Scientist:

Our Microsequencing technology relies on two basic criteria that require specific sample conditions that are as follows:

1. Your sample must be immobilized on PVDF membranes:

The primary reason for this is ease of shipping and handling. There are several methods to immobilize on PVDF:

SDS-PAGE and electrotransfer , spin cartridges, and adsorption methods (please call for more details). Once your protein/peptide is on PVDF and dried it is reasonably indestructible (and we know where it is). The stained band is simply cut from the PVDF that actually shows visible staining, if the band "smiles", cut out only the "smile". Microsequencing becomes much more inefficient when sequencing empty PVDF. If your band is only 0.5mm x 3mm -as from mini gels-then we expect to receive a band cut out that is 0.5mm x 3mm only! Coomassie and other stains fade quickly with drying so it is reasonable for you to excise the band precisely with a fresh razor blade while the band is fresh.

After excising the band, air dry on the bench top for approximately 30 minutes at room temperature. The band may be stored frozen until ready to ship. We have sequenced bands frozen for up to six months without substantial loss in n-terminal signal. The band should be placed in a 1.5ml Eppendorf (R) type micro tube and shipped to us via overnight delivery (FedX or equivalent). There is no need for ice packs or refrigeration of the sample during the overnight delivery.

We can obtain good sequence information using any brand of PVDF however we prefer second generation small pore PVDF (0.1 to 0.2 micron) offered by Applied Biosystems (ProBlott [R]), Millipore (Immobilon-PSQ [R]), or BioRad (TransBlott [R]). The advantage to you is realized in electroblotting where it is virtually impossible for protein to completely pass through these membranes and become lost in the transfer buffers. The advantage to us on the sequencing end is the tendency of proteins to be held tighter by second generation PVDF. Note: 0.1 to 0.2 micron PVDF is required for internal sequencing.

2. Your sample must be visible by Coomassie stain:

A general rule to keep in mind is that where possible, try to obtain the maximum concentration of protein on the smallest area of PVDF.

One reason that mini gels sequence well is that even though there are only nanogram quantities of protein, this protein is concentrated on PVDF only a few square millimeters in size! For sequencing purposes it is far more advantageous to supply us with a single band of concentrated protein rather than several bands of very dilute protein on PVDF.

A typical band for low microgram quantities of protein should be 2mm x 10mm or smaller for best sequencing results. If you can visualize the band well enough to excise it, then there should be sufficient concentration for sequencing.

If you have technical questions please write proseq@tiac.net

Or Call (978) 213-9911 or fax (978) 213-9922

For International callers please add the USA country code

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	Sample Requirements: MICROSEQUENCING ON PVDF MEMBRANES Dear Scientist: Our Microsequencing technology relies on two basic criteria that require specific sample conditions that are as follows: 1. Your sample must be immobilized on PVDF membranes: The primary reason for this is ease of shipping and handling. There are several methods to immobilize on PVDF: SDS-PAGE and electrotransfer , spin cartridges, and adsorption methods (please call for more details). Once your protein/peptide is on PVDF and dried it is reasonably indestructible (and we know where it is). The stained band is simply cut from the PVDF that actually shows visible staining, if the band "smiles", cut out only the "smile". Microsequencing becomes much more inefficient when sequencing empty PVDF. If your band is only 0.5mm x 3mm -as from mini gels-then we expect to receive a band cut out that is 0.5mm x 3mm only! Coomassie and other stains fade quickly with drying so it is reasonable for you to excise the band precisely with a fresh razor blade while the band is fresh. After excising the band, air dry on the bench top for approximately 30 minutes at room temperature. The band may be stored frozen until ready to ship. We have sequenced bands frozen for up to six months without substantial loss in n-terminal signal. The band should be placed in a 1.5ml Eppendorf (R) type micro tube and shipped to us via overnight delivery (FedX or equivalent). There is no need for ice packs or refrigeration of the sample during the overnight delivery. We can obtain good sequence information using any brand of PVDF however we prefer second generation small pore PVDF (0.1 to 0.2 micron) offered by Applied Biosystems (ProBlott [R]), Millipore (Immobilon-PSQ [R]), or BioRad (TransBlott [R]). The advantage to you is realized in electroblotting where it is virtually impossible for protein to completely pass through these membranes and become lost in the transfer buffers. The advantage to us on the sequencing end is the tendency of proteins to be held tighter by second generation PVDF. Note: 0.1 to 0.2 micron PVDF is required for internal sequencing. 2. Your sample must be visible by Coomassie stain: A general rule to keep in mind is that where possible, try to obtain the maximum concentration of protein on the smallest area of PVDF. One reason that mini gels sequence well is that even though there are only nanogram quantities of protein, this protein is concentrated on PVDF only a few square millimeters in size! For sequencing purposes it is far more advantageous to supply us with a single band of concentrated protein rather than several bands of very dilute protein on PVDF. A typical band for low microgram quantities of protein should be 2mm x 10mm or smaller for best sequencing results. If you can visualize the band well enough to excise it, then there should be sufficient concentration for sequencing. If you have technical questions please write proseq@tiac.net Or Call (978) 213-9911 or fax (978) 213-9922 For International callers please add the USA country code