Protocol tips
Upstream tips |
Protocol tips |
Downstream tips |
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year. |
-Rabbit (1:1000)
-To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for
5 min. Lysates can be aliquoted and stored at -20°C for future use.
|
|
Upstream tips |
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year. |
Protocol tips |
-Rabbit (1:1000)
-To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for
5 min. Lysates can be aliquoted and stored at -20°C for future use.
|
Publication protocol
n our experience, the membrane blocking agent is very important in Western Blot assay. In the Western Blot assays, we selected the skim milk agent from BD Co. for blocking. Usually, Western Blot assay is used for qualitative detection of expressed proteins. We are grateful to the review’s suggestion for increase of controls that will make our evidence more solid. Light chain of botulismotoxin D (BoNT/D-LC) or botulismotoxin C (BoNT/C-LC) has been reported as the VAMP2 or SYN1 cleaver (Rossetto et al., 2014). According to the reviewer’s request, we added the controls for the expression and staining of SYN1 in the BoNT/D-LC-transfected cells and VAMP2 in the BoNT/C-LC-trabnsfected cells in the Supplementary Information of revised manuscript. The results showed that the BoNT/D-LC or BoNT/C-LC transfection did not affect the expression and fluorescence staining of SYN1 or VAMP2.
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Manufacturer protocol
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