Immunoprecipitation Materials

This example shows that immunochemical methods are not neccessarily highly specific. Cross reactions with immunoprecipitable material or solubilization of antigen complexes can falsify the assay to a considerable extent. Currently more sophisticated techniques bearing a higher specifity than the older methods are employed They are used in many laboratories for routine purposes The most reliable... 

The concentration of protein in the lysate should be determined as a guide to even loading of gels or the amount of material to be subjected, for example, to immunoprecipitation. A simple and reliable method for this is that of Bradford (Bradford, 1976). 

Hanna PA, Jankovic J, Vincent A (1999) Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies. J Neurol Neurosurg Psychiatry 66 612-16 Hanson MA, Stevens RC (2000) Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution. Nat Struct Biol 7 687-92 Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog s neuromuscular junction. Nature 409 479-84 Harris JB (1997) Toxic phospholipases in snake venom an introductory review. Symp. zool. Soc. Lond. 70 235-50... 

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

The polyclonal antiserum, obtained by immunisation of rabbits as described in Materials and Methods was initially used for immunoprecipitation of metabolically labelled primary cortical neurons, providing a major band at 110 kDa corresponding to intact neuronal APP (Figure 2 A). The antibody was also tested by western analysis yielding... 

Both phases are then diluted to 1 ml with PBS (0 "C), followed by immunoprecipitation with antibodies adsorbed to protein A-Sepharose. The precipitated material is analyzed with SDS-PAGE and tiuorography. 

Fig. 3. Purification of complexes assembled in vivo. Depending on the material available, classical biochemical purihcation (left), co-immunoprecipitation (center), or affinity purihcation (right) may be applied. The advantages and inconveniences of the various strategies are described in the text. The material purihed using these various strategies is characterized in the same way.

The involvement of the proteasome suggests that the MHC class I heavy chains migrate back to the cytosol. Indeed, a cytosolic disposition of the deglycosylated intermediate can be confirmed by subcellular fractionation experiments. Furthermore, this intermediate can be immunoprecipitated from material reactive with anti-proteasome antibodies. Apparently, US2 and USll induce retrograde translocation, or dislocation , of MHC class I heavy chains from the ER to the cytosol, where the heavy chains are subjected to proteolytic degradation by the proteasome (Wiertz et al. 1996a,b). 

To immunoprecipitate proteins from embryonic tissue or cell culture, the material has to be disrupted under conditions that solubilize the proteins (see Notes 3 and 4). 

Immunoprecipitation is a technique that allows the biochemical characterization of protein from relatively few cells, and is an important technique where material may be a limiting factor. It can also be used to study various types of posttranslational modification (see Note 8). The amount of material required will depend on the abundance of the antigen (see Note 9). 

Use 5-25 ng of immunoprecipitated DNA to prepare the library. Run an aliquot of the ChIP DNA on an Agilent Technologies 2100 Bioanalyzer using a high sensitivity DNA chip to verify the size and concentration of the DNA. If necessary, material ftom several immunoprecipitations may be pooled to obtain the required quantity (at least 5 ng) of starting DNA. 

Quantitative immunoprecipitation experiments were performed to determine the relationship between enzyme activity and immuno-precipitable material for purified rat liver, N1S167 (sensitive parent) and 5-2 (resistant cell) ADA s. Figure 1 shows that the same amount of antibody precipitated the same amount of enzymatic activity for all three proteins. Thus, ADA s from rat liver and dCF sensitive hepatoma and resistant hepatoma cells are indistinguishable in terms of kinetic, physical and immunological properties. The increase in ADA activity in dCF cells is clearly due to an increase in the number of molecules of a structurally normal enz3nme. 

---