Western Blot Dot Blots

Last updated: 1/12/12 By: Tim Starr

Overview

Instead of running protein sample on gel and transfering to membrane, you can just place a drop of protein directly onto the membrane. Do this to test whether your antibodies can detect the protein. The following protocol is taken from the Qiagen Bench Guide.

Protocol

• Dilute protein samples in dilution buffer to final protein concentration of 1 to 100 ng/µl.
• Place 1 µl on a dry membrane (Qiagen recommends nitrocellulose).
• Let the sample dry for a short time at room temperature.
• Alternatively, can apply crude cell lysate (1 µl) if you think the concentration is 1 to 100µl ( I assume this is the concentration of the protein you wish to detect, not total protein concentration).
• Follow protocol for detecting proteins using antibodies

Dilution buffer 1 x, 1 L (denaturing conditions) adjust pH to 8.0