Blocking reagents recommendations

In practice, including blocking reagents in all incubation and rinse solutions insure that the tissue is adequately blocked. It is recommended that the blocking regents be added to all subsequent incubation steps, and if possible, to the rinse steps between incubations. 

In 1966 Block et al. (B3, B4) published a modification of the Zlatkis et al. and Levine and Zak technics. Manually prepared serum extracts are added to a stream of preheated color reagent, and the mixture is then passed through three mixing coils connected in series. The absorbance is determined at 550 mju, using a 15-mm tubular fiow cell. This technique is currently recommended as the total cholesterol procedure by the Technicon Instruments Corp. (C4, C5). 

Fmoc amino acid fluorides, highly carboxy-activated amino acid derivatives suited both for solution syntheses and for SPPS. These species are especially recommended for SPPS of complicated longer peptides, and also mainly for the coupling of steri-cally hindered amino acid building blocks. Fmoc amino acid fluorides are usually stable crystalline derivatives and can be synthesized from the Fmoc amino acids with DAST, cyanur fluoride, or the TFFH reagent [L. A. Carpino et al., J. Org. Chem. 1986, 51, 3732 L. A. Carpino et al, J. Am. Chem. Soc. 1990, 112, 9651 L. A. Carpino et al., J. Am. Chem. Soc. 1995, 117, 5401]. 

As already stated, whole serum can be used to coat plates and act as a capture reagent. However, this is not recommended because we cannot measure the protein Ig because it is contaminated with "blocking" serum proteins that compete for plastic binding sites preferentially over the IgG. The simplest method is to perform a CBT relating dilutions of capture serum to dilutions of detecting antibody and keep the antigen constant. 

Should this profile be distorted it may indicate that there is a problem with the smooth and rapid flow of the reagents, possibly syringes sticking or the viscosity of the solutions not permitting the solutions to accelerate fully to the desired velocity. Alternatively the pressure is low. Check the pneumatic pressure, clean the system as recommended. Possibly the apparatus needs to be adjusted to deliver more of each solution by moving the. stopping block. 

The lithium enolate of (R)- or (S)-l-benzoyl-2-ferf-butyl-3-methylimidazolidin-4-one (Seebach s reagent, 229), for example, furnishes the respective primary aldol adducts in excellent diastereoselectivities (d.e. >90%). Their hydrolysis to the free acids, however, requires such drastic reaction conditions (6N HCI, 150 °C, sealed tube) that these building blocks are of very limited use. For this reason the corresponding (R)- and (5)-l,3-oxazolidin-5-ones 230 are recommended as more useful alternatives. In their case hydrolysis of the primary adducts can be accomplished under very mild reaction conditions, namely hydrogenolytic removal of benzyloxycarbonyl function followed by hydrolytic cleavage of the heterocyclic system with LiOH/aq. THF or IN HCI at room temperature. ... 

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