QuestionScenario 3 You are about to do a Western Blot transf

Question/Scenario 3:

You are about to do a Western Blot transfer. You have two identical transfer membranes, but are unsure what kind they are. Following your standard protocol, you dip one of the membranes in100% methanol, and it instantly dissolves. Your attempts to remove it from the alcohol just break it into thousands of wispy fragments. Your gel is ready to transfer now, and you only have one membrane left. You don’t have time to go buy more. What can be done?

Solution

The standard procedure of Western blot has a step for dipping the membrane in 100% methanol. PVDF membrane being more hydrophobic in nature than nitro-cellulose membrane endures this step successfully. But often nitrocellulose membranes are found to be more brittle and disintegrate while soaking in 100% methanol. Thus the methanol needs to be diluted and a transfer buffer needs to be used for membrane soaking. Ideally 20% methanol and tris-glycine buffer (1% transfer buffer) should be used for the 2nd membrane.