Automation protocols manual methods

The primary antibody imparts specificity to the IHC method, and may be obtained either as an antibody concentrate, or as an RTU reagent. If obtained as a concentrate it must be adjusted to an optimal working dilution for use with a secondary labeling system selected by the laboratory. If obtained as an RTU reagent, it typically is purchased along with an optimized labeling system and defined protocol, for use with an automated Stainer, or sometimes by manual methods. 

For systems that do not offer online HIAR, these procedures must be performed manually or off-line prior to loading slides on the instrument. Online HIAR methods are usually found as part of the closed-type automated staining systems, and are therefore less flexible in terms of what a technician can do to change the HIAR portion of a staining protocol to help optimize staining for particularly difficult antibodies. In spite of this, the ability to perform online HIAR is advantageous for many antibodies because it is extremely consistent and frees up technician time to complete other laboratory tasks. 

The inhibitory potencies of three of these molecules agreed with published values for murine Kv 3 (Table 4.1). The one exception was ShK that appeared to be 10-fold less potent than both published values and our own in-house values obtained by manual patch-clamp methods. It has been reported that ShK has a slow on-rate (t= 20 min) for block of Kv 3 (Middleton et al. 2003). This phenomenon may contribute to the reduced potency of ShK in our automated electrophysiology assay, because our protocol included a compound incubation time of only 5 to 10 min. Longer compound incubation times may improve the potency of ShK but would be associated with greater run-down in the K+ current amplitude. We also tested the A LI-selective blocker, dendrotoxin, and not surprisingly it did not inhibit the Kv. 3 current at concentrations up to 167 nM, which is well above its IC50 value for Kv 1.1 in our hands (17 pM data not shown). 

Working on actual serum samples, Wang et al. (2004a) measured morning serum T concentrations in samples from 62 eugonadal and 60 hypogonadal men (25 baseline samples, 35 after transdermal T replacement therapy) in order to compare a reference method by LC-MS-MS with six commonly used immunoassays, four or them automated and two manual. In this smdy, the analyte was T, therefore it was possible to use 6Z3-T as internal standard for LC-MS-MS analysis. The method was validated using protocols specified by the... 

The protocols have been written as they would be carried out using a manual peptide synthesis vessel. Whilst it is appreciated that most scientists preparing peptides will be using automated peptide synthesizers, it is not possible, given the wide variation in operating procedures, to describe how such methods may be applied to individual instruments. Particular emphasis has been given here to those operations which are typically carried out off-instrument, such as first residue attachment and peptide-resin cleavage. 

This protocol outlines the general procedure for both manual and automated microwave-assisted peptide synthesis. For the manual synthesis the acyl carrier protein, AGP (65-74) sequence was selected as a representative difficult peptide. The automated synthesis features the preparation of p-amyloid (1 2), another peptide that is known to be difficult to synthesize. Both methods provide the peptides in excellent purity with good yields in a fraction of the time it would take to perform these syntheses using conventional, room temperature synthesis conditions. 

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