Goat antirabbit IgG

For detection of Fab antibody fragments, use a polyclonal rabbit IgG to human k and X light chains and HRP-conjugated goat antirabbit IgG (see Note 9). 

Antiserum at 37°, 15 min Wash 0°. +Lectin 100-300 yg/ml at 0°, 30 min Wash, 0° Rabbit anti-lectin serum, 0°, 15 min Wash 0° Fluorescein labelled goat antirabbit IgG, 0°, 15 min Wash 0° View. FITC, Fluorescein isothiocyanate. TABLE II. Inhibition of Immunofluorescence ... 

Goat-antirabbit IgG (HRP conjugated) (Kirkegaard Perry product 074-1506)... 

Use goat-antirabbit IgG secondary antibody for rows A to D. Use goat-antimouse IgG secondary antibody for rows E to H. 

HRP-conjugated-goat-antirabbit IgG —Each group will prepare 2.5 ml of this solution by making serial dilutions of the stock antibody (Kirkegaard Perry catalog 074-1506) in blocking solution. The final dilution should be about 1 9000. Prepare immediately before use. 

Immunoglobulin type, reactivity with chemically modified antigens, and carbohydrate content of antibodies may also be determined. In determination of the Ig type it was found that all antibodies react with goat antirabbit IgG and not against rabbit IgA or IgM. The antibodies are therefore of the IgG type of immunoglobulin.37 Periodate oxidation, oxidation by... 

Secondary antibody (e.g., goat antirabbit IgG-peroxidase conjugated if primary antiserum is from rabbit). 

Fig. 1. An immuno-slot blot fin the detection of 0 -hydro]Q thy]deoxygiianosine. Calf thymus DNA was hydrm ethylated in vitro with 6.6 mAf of hydroxyethyl-nitrosourea. Aliquots containing 300, 100, 33.3, 11.1, 3.7, 1.2, and 0 finol of O -hydroxyethyldeoxyguanosine in 3 (ig ofheat-denatured DNA (corresponding to 217-0 pmol/tnol of deoxyguanosine) were blotted onto nitrocellulose and incubated with a rabbit anti-O -hydroxyethyldeoiQ guanosine serum (NPZ-146-2,1 15,000 see ref. 4). Bound antibodies were reacted with a goat-antirabbit IgG horseradish peroxidase conjugate and detected with hydrogen peroxide and 4-chloro-l-naphth6l.

Fig. 2. Typical densitometnc scan. Calf thymus DNA, methylated in vitro with 5 mM of methylnitrosourea, was denatured in 50 mM sodium hydroxide and blotted onto nitrocellulose. After incubation with a rabbit antiserum specific for O -methyldeoiQrguanosine (NPZ-193-1,1 8,000 see ref. 4) and alkaline phosphatase conjugated goat-antirabbit IgG, the blot was developed with 5-bromo-4-chloro-3-indolylphosphate toluidine salt and nitroblue tetrazolium chloride in diethanolamine buffer, as described in the Methods section, and scanned by reflectance densitometry at 530 nm. Slots contain 283, 94.7, 31.3, 10.4, 3.4, 1.1, and 0 fmol of 0 -methyl-deoxyguanosine, corresponding to 181-0.7 pmol/mol of deoxyguanosine.

Treatment with 2% horse USERIA serum incubation with rabbit anti-BPDE-DNA antiserum incubation with alkaline phosphatase-conjugated goat antirabbit IgG. PNPP, radiolabeled PNPP, and MgClg separation of hydrolyzed radiolabeled PNPP measurement of radioactivity... 

First, antibodies against the protein to be measured are developed in a host animal. For example, protein can be the human cardiac troponin-T (h-cT), which is a marker of myocardial infarction. Purified h-cT is injected into a rabbit to raise IgG molecules against h-cT, and these antibodies can either be recovered from the blood or produced recombinantly in a bioprocessor. A secondary antibody is also needed, which reacts with the first antibody and provides a colored solution. For example, this secondary antibody can be the goat antirabbit IgG antibody, which is tagged with a coloring compound or a fluorescent molecule. This second antibody can be used for any ELISA test that utilizes rabbit IgGs, regardless of the analyte, and such secondary antibodies are usually available commercially [6]. 

If amplification is required, apply 50 ju,l biotinylated sheep antiavidin antibodies (1 200 in buffer C) and 50 1 rabbit antimouse IgG antibodies (1 200 in buffer C) and incubate for 1 hr at 37°C under a cover slip. Wash 3x with buffer C at 37°C. Apply 50 /x.1 of FITC-conjugated ExtrAvidin (as above) and 50 ml TRITC-conjugated goat antirabbit IgG antibodies (1 200 in buffer C) and repeat the incubation step. Finally, wash the slides 3x in buffer C as above, shake off the liquid, apply a drop of antifade medium plus DAPI (see above) under a cover slip, and seal with rubber cement. 

Biotinylated goat antirabbit IgG (2° antibody Vectastain Elite ABC kit Vector Laboratories, Burlingame, CA, PK6101). 

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