How are the data from SPOT synthesis evaluated? Detection is either by enzymatic color deposition and simple densitometry using a flatbed scanner, or by exposure of a film if e.g. radioactive phosphorus has been incorporated. Detection and data handling are standard techniques which we do not support.
What is the approximate cost of SPOT synthesis? The cost of SPOT synthesis on cellulose membranes can be estimated from the number of membranes per synthesis. It is largely unrelated to the actual number of peptides, as the consumption of amino acid derivative solution per spot decreases with the spot size as the number of peptides per membrane increases. The amount of solvent for bulk treatment is completely unrelated to the number of peptides per membrane. A calculation for 4 membranes, 4x 96 or 4x 384 peptides of 10 residues yields a total cost of approximately two times the cost of derivatized membranes as INTAVIS or our representatives supply them.
Which chemicals are provided by INTAVIS? We only offer Fmoc amino acids in vials of 0.5 mmoles, each. They are to be activated by the DIC/HOBt method. Other chemicals, such as activator, solvents and reagents must be acquired from a local source. We provide a list of chemicals needed and sources for them.
What is the time for SPOT synthesis? For a synthesis of 4x384 peptides with a double coupling protocol you have to expect a duration of 2 hours per cycle and 2-3 hours for final work up. The hands-on time per cycle is about 30 minutes for preparation of activated derivatives and washing of the membranes.
What is the density of peptides on the membrane? The standard density is a grid of 96 peptides in microtiter plate spacing or a grid of 384 spots with 4.5 mm center-to-center distances. Other grids are possible and can be freely defined up to a number of 10 grids. Densities of up to 25x40 spots on a cellulose membrane have been achieved under very controlled conditions.
How is the instrument spotting reliability (volume and position) validated? How is the peptide synthesis reliability (% purity, amount) validated? There is no validation except for comparison with reference sequences to be defined by the user for his respective assay. It is very difficult to check the purity and quantity. There are a few literature references to procedures used. In general, you should work out internal references and standards for your assay.
What are the chemistry limitations regarding the peptide length? We recommend peptide lengths of no more than 10-12 residues. It has been shown that the synthesis quality drops sharply after a chain length of 8-10 residues, probably due to sterical reasons. Due to the open synthesis conditions severe oxidation of cysteine and methionine must be expected.
What is the chemical stability of the membranes? The membranes used are based on acid hardened cellulose and derivatized with a polyethylene glycol spacer. This spacer is stable in a pH range of 1-14. The paper itself is stable to the synthesis conditions but should not be exposed to strong acid for prolonged periods. The cleavage by dilute trifluoroacetic acid for 1-2 hours is the limit of chemical stability.
Are peptides lost by repeated stripping? The original protocol published is based on derivatization of the paper with beta-alanine by an ester bond. This bond was cleaved under slightly basic conditions, e.g. most of the peptides would be lost over the course of some hours at pH 8. The new PEG spacer introduced in 1998 eliminates this problem. There may still be users who make the derivatized membranes themselves according to the old published protocols which will create the problems mentioned.
What is the loading of the membranes? The membranes we supply are derivatized to a loading of about 300-400 nmol/sqcm free amino groups on PEG spacers of about 500 Da molecular weight.
