Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.
Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.
Ambroz
Kristi L H
KL
LI-COR Biosciences, 4647 Superior Street Lincoln, NE 68504, USA. kristi.ambroz@licor.com
Zhang
Yonghong
Y
Schutz-Geschwender
Amy
A
Olive
D Michael
DM
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D019138:Antibodies, Blocking / Q000737:chemistry*; D000918:Antibody Specificity; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D005453:Fluorescence; D019084:Fluorescent Antibody Technique, Indirect; D007202:Indicators and Reagents / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D049449:Luminescence; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared; D019809:Streptavidin / Q000737:chemistry; D014439:Tyramine / Q000737:chemistry
D000906:Antibodies; D019138:Antibodies, Blocking; C427728:IRDye800; D007202:Indicators and Reagents; D007211:Indoles; D001710:Biotin; D019809:Streptavidin; D014439:Tyramine