I have two questions:
1. After transfer, the marker confirmed that the transfer had proceeded well. However, I would like to know why only one protein band appears in the high kDa region when ponceau S staining is performed.
2. I would like to know why the protein band I want to check cannot be identified during detection and why chemiluminescence occurs at the bottom of the marker. (This is a case where a problem occurred that did not exist before.)
Condition :
running 110V 110min
transfer 230mA 90min
membrane : NC membrane 0.2um
blocking : 5% skim milk RT 2hr
primary antibody : V5-tag (Rb) 1:1000 5% skim milk
secondary antibody : Rabbit-IgG 1:4000 5% skim milk
Detection :
using
SuperSignal™ ELISA Pico Chemiluminescent Substrate (Not detected)
SuperSignal™ West Femto Maximum Sensitivity Substrate (Not detected)
Fusion fx vilber lourmat (western blot and chemiluminescence imaging system)
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