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Thank you very much for your information. If I am transferring a small protein molecule around 3 kDa MW, what transfer condition would you recommend me to use in terms of voltage and time of transfer using this membrane? Thank you very much for your time,
Asked on Nov 21 2012
Thank you for contacting us.
Generally when transferring a very small protein, it is best to use a semi-dry transfer procedure. These usually run at 100V for 40-60 minutes. I would recommend transfer times at the lower end to this range. It is also helpful to remove SDS from your transfer buffer when transferring small proteins. I have included with this email our Western blot guide which includes further transfer information. Below I have included a link to our WB reagents page which has a semi-dry transfer buffer recipe. Finally, I have a brief protocol for semi-dry transfer which I will place at the end of this email.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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1. Cut 6-10 sheets of filter paper same size as the gel.
2. Cut membrane to the size of the gel.
3. Membrane activation for PVDF: soak in methanol for 15 seconds and then transfer to a containerfilled with distilled water for 5 minutes.
4. Soak membrane in semi-dry transfer buffer for 10 minutes while preparing transfer sandwich
5. Soak filter paper in transfer buffer for several minutes to 10 minutes to avoid trapping of air bubble.
6. Assemble sandwich in the following order:
3-5 sheets filter paper -
3-5 sheets filter paper +
7. Use a roller to roll over the sandwich gently to remove trapped air bubbles.
8. Apply few ml transfer buffer on top of the sandwich to avoid drying out during membrane transformation.
9. Transfer at 100 V for 40-60 minutes.
10. Block and Stain the membrane as normal
Answered on Nov 21 2012