Dear all,
I am now investigating the expression of small size protein (6 kda). However, the expression of the protein cannot be shown clearly after performing western blot. Therefore, are there any methods that can help me for protein detection?
Ting Chung Chau
you can try ELISA especially if you have Tag-protein
Hello Ting Chung Chau
Besides Western Blot you can detect protein spectrometrically by liquid chromatography-mass spectrometry (LC-MS) or high performance liquid chromatography (HPLC).
There are also methods which are antibody dependent such as Western Blot, dot blot, ELISA and protein immunoprecipitation.
Good Luck.
Such a small protein could be transferring through your membrane if you transfer for too long and/or at high voltage. If you have an established western blot protocol, I would play around with transfer steps; some suggest 10-15 minutes at 200mA. To be sure, I would probe against other small proteins as a positive control in order to exclude the possibility that your antibody isn't targeting your protein of interest.
Another possibility is that your gel is too low in acrylamide. Resolving a 6kDa protein requires a relatively high acrylamide proportion, maybe around 15-20% acrylamide.
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