The most common practice when performing a western blot is to use a secondary antibody to detect the proteins of interest. This is advantageous if there is a need to amplify the signal. However, if the target under study is abundant, and the quality of a directly conjugated primary antibody to Horse Radish Peroxidase (HRP) is high, there is really no advantage or need to use a secondary conjugated antibody. Thus, we are happy to introduce Direct-Blot™, primary antibodies directly conjugated to HRP, to help you speed up, simplify and reduce the cost of your experiment.

 

Summary of advantages:

  • Shorter protocol
  • Highly sensitive
  • Excellent stability
  • Easier to multiplex
  • Easier to combine with Immunoprecipitation
  • No potential background from HRP conjugated secondary antibodies

 

Sensitivity

15 µg of total protein from either HeLa cells or Jurkat cells treated with PMA (anti-phospho ERK) was resolved by electrophoresis, transferred to nitrocellulose, and probed with different concentrations of HRP directly conjugated antibodies (Direct-Blot™) or purified monoclonal (Pur) anti-GAPDH (clone FF26A/F9 ), anti-Vimentin (clone O91D3), anti-ERK1/2 Phospho (Thr202/Tyr204) (clone 4B11B69) antibodies at a fixed concentration (0.5 µg/mL). For Direct-Blot™ antibodies, proteins were visualized using chemiluminescence detection directly. For purified antibody, proteins were visualized using a goat anti-mouse-IgG secondary antibody conjugated to HRP (Cat. No. 405306) followed by chemiluminescence detection.

Stability

 

10 µg of total protein was resolved by electrophoresis and transferred to a PVDF membrane. HRP directly conjugated (Direct-Blot™) antibodies were aliquoted and kept at 4°C, RT and 37° C for 2, 4, 7, and 14 days. Graphs show the percentage of activity of anti-GAPDH and anti-Vimentin at room temperature and 37°C, expressed as percentage of activity at 4°C.
The band intensity quantification was done using the ImageJ software.

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