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Western Blotting Application Notes

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Antibody Reuse: Antibody Recovery and Reuse with the SNAP i.d.® Protein Detection System

Recovering the primary antibody for possible reuse can be very valuable for Western blotting experiments. Here, we show that greater than 90% of the primary antibody can be recovered after the incubation step by following the recommended protocol. The antibodies collected in the SNAP i.d.® 2.0 System can be used successfully in subsequent immunodetection with no reduction in blot quality, even after extended incubations.
Antibody Recovery and Reuse With the SNAP i.d.® Protein Detection System
Comparison of cell lysate blots probed with fresh and reused antibodies using the SNAP i.d.® 2.0 system.The blots were probed with anti-PP2A, which was either freshly diluted or recovered and reused, following the SNAP i.d.® 2.0 system extended protocol. Click image to enlarge.

Immunodetection: Key Steps for Successful Immunodetection Using the SNAP i.d.® Protein Detection System

The Western blot is a powerful tool used extensively in protein research to detect and compare the relative levels of proteins without the need for their prior purification. Its widespread appeal is based on its overall simplicity, coupled with the high resolution of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) prior to membrane transfer.
Key Steps for Successful Immunodetection Using the SNAP i.d.® Protein Detection System
The SNAP i.d.® Protein Detection System has been developed to shorten the time required for immunodetection (i.e., blocking, washing and antibody incubations) to approximately 30 minutes. Click image to enlarge.

Fluorescent Immunodetection: Fluorescent Immunodetection Using the SNAP i.d.® Protein Detection System

Recent advances in fluorescent dye chemistry and blotting membranes coupled with improvements in instrumentation have accelerated the application of fluorescence detection methodologies to Western blotting. The SNAP i.d.® Protein Detection System has been developed to substantially shorten the time required for fluorescent immunodetection through the use of vacuum.
Fluorescent Immunodetection Using the SNAP i.d.® Protein Detection System
Cy3 fluorescent immunodetection of GAP43 in human brain lysate. The blot was visualized using Fujifilm FLA_5100 imaging system and was quantified with an R2 of 0.98. Click image to enlarge.

Evaluation of Common Protein Extraction Reagents in Mammalian and Bacteria Lysates, by Infrared (IR) Based Quantification and Western Blotting

The quality of sample preparation ultimately impacts the quality of the downstream Western blot. For example, if conditions are too aggressive, the marker may be denatured or destroyed. If not sufficiently stringent, the marker may be lost in the insoluble fraction. We evaluated several buffers and reagents commonly used for protein extraction from mammalian and bacterial lysates. An accurate determination of total protein concentration allowed well-resolved protein separation by electrophoresis followed by Western blotting analysis of some of the proteins using a new, rapid Western blotting method.
Total protein content liberated by BugBuster® Extraction Reagent was much higher than the amount produced by homebrew method. Also, the addition of Benzonase® Nuclease and rLysozyme™ solutions had a significant impact on overall yield. E. coli lysates from various lysis protocols were fractionated and analyzed by SDS-PAGE. A band corresponding to 6XHIS-CRP is prominently visualized in the BB +/+ lane.