1° antibody controls immunoblot

Fig. 2. Tissue-print immunoblots of cross sections from okrafruitpods. Ponceau S protein staining of blots before immunodetection reveals the anatomical detail of sections subsequently probed with control serum (A) or with Phaseolus vulgaris IAA-peptide antibody (B). Arrows indicate the position of seeds remaining within the cross-sectioned pods.

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Precise controls for the specificity of antibody reactions reside with other techniques, such as immunoprecipitation or immunoblotting. Controls for the labeling... 

Fig. 1.2 Effect of in vivo PT treatment on the levels of Gia-2 and Gioc.-3 proteins. Upper panels Heart membrane proteins (20 pg) from 6- and 8-week-old SHR and WKY with or without PT treatment (Li and Anand-Srivastava 2002) were resolved by SDS/PAGE and transferred to nitrocellulose, which was then immunoblotted with antibody AS/7 for Gia-2 (A) or antibody EC/2 for Gia-3 (B). The blots are representative of three or four separate experiments. Lower panels Quantification of protein bands by densitometric scanning. The results are expressed as percent of WKY control at 6 weeks, which has been taken as 100%. Values are means S.E.M. of three or four separate experiments. Reproduced with permission from Li and Anand-Srivastava (2002).

Fig. 1.5 Effect of iV-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI) on Gia-2 and Gioc-3 protein expression in vascular smooth muscle cells (VSMC) from 12 week-old SHR and age-matched WKY rats. Confluent VSMC from SHR and WKY rats were treated with 20 mM NAC (A) or 10 pM DPI (B) for 24 hours at 37 °C. Membrane proteins (30 pg) were separated and transferred to nitrocellulose, which was immunoblotted with antibodies AS/7 and EC/1 against Gioc-2 (A) and Gioc-3 (B), respectively, as described earlier (Lappas et al. 2005). The blots are representative of five separate experiments. The graphs in the lower panel show quantification of protein bands by densitometric scanning. The results are expressed as percentage of WKY control which has been taken as 100%. Values are mean S.E.M of five separate experiments. P < 0.05 versus WKY, < 0.01 versus SHR.

Hamilton, R. G., Reimer, C. B., and Rodkey, L. S. (1987). Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis. Hybridoma 6, 205-217. 

Fig. 3. Association of TrkA receptor with p75 in PC 12 cells. TrkA-overexpressing PC12 (615) cells were treated with or without NGF (50 ng/ml, 10 min) and then harvested in TNE buffer. Cell lysates (18 mg of protein) were immunoprecipitated with either anti-pan Trk IgG or normal IgG as a negative control (as described - or - on top of the panel). The resulting immunocomplexes were analyzed by immunoblotting with anti-p75 antiserum (9992). The immunoprecipitation of TrkA was confirmed by immunoblotting of a portion of the immunocomplex with anti-pan Trk serum and reprobed with anti-phosphotyrosine antibody (pY99, Santa Cruz). The amount of p75 protein in each lysate was analyzed by western blot analysis on the two rightmost lanes of the top panel.

Most commonly used controls are immunoblots with the tissue of interest to determine whether the 1° antibody can bind to a single protein of the correct molecular weight. 

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