Have you ever wished you could transfer the same SDS-PAGE gel twice? Sometimes, when you are blotting for many different proteins of similar size, stripping and reprobing multiple times can become impractical. Here’s a simple diffusion transfer method that can be used to generate duplicate membranes from a single gel:
- Take a glass plate, or something that is similarly sturdy and flat.
- Layer the following onto the glass plate in order, smoothing each layer out to remove air bubbles:
- Three pieces of blotting paper (e.g. Whatman 3MM) that have been soaked in phosphate-buffered saline (PBS).
- Lay out a pre-soaked membrane (I’ve only tested nitrocellulose, but PVDF should be fine).
- Your gel.
- Another pre-soaked membrane.
- Three more pieces of PBS-soaked blotting paper.
- Add another glass plate.
- Tightly wrap the whole sandwich in plastic wrap.
- Place a heavy object (e.g. a 1L bottle of buffer) on top.
- Go home for the night, or for the weekend.
Protein transfer by this diffusion method is much slower than electrotransfer, but if you give it enough time, it is surprisingly efficient. However, it is not recommended for very large proteins. Also, don’t forget that you will have about half as much protein on each membrane compared to transferring to only a single membrane.
Try this method out, and let us know how it goes! It sounds too low-tech to work, but the few times that I’ve tried it has given me great results.
Excellent idea, Cristy!
One thing that seemed to be omitted was some sort of soaking. Is the pre-soaked filter paper sufficient to keep the membrane from drying out overnight?
Also, do you have some results that demonstrate the efficacy of the method? Intuitively, it seems one might get better transfer to the membrane at the bottom of the sandwich.
Thank you! I would love to try this out!
Hi,
The sandwich won’t dry out, as long as you wrap it up well with plastic wrap (and make sure all the filter paper is well saturated). It doesn’t seem to me that I get better transfer at the bottom of the sandwich, but I’m not sure I would trust the method to give identical efficiency to each membrane. That being said, I would say the same about the alternative, which is to run two gels and transfer them separately, and the important thing is that the transfer is even across the gel. Hope it works well for you!
This is actually an “old school” transfer method that was the first blotting technique I ever learned, as a grad student! Works fine, the only disadvantage is the longer overnight waiting time. On the other hand, most people incubate the membranes with the antibody overnight, whilst this technique allows you to do all the incubations and development on the same day, right after the transfer.
Also, it feels liberating to leave a transfer over the weekend!
Great method, but you can do it even faster (as short as 7 minutes) and to multiple membranes if you leave the blotting paper dry (Check out Kurien and Scofield, Journal of Immunological Methods 2051997.91–94).
This method also gets around the pesky problem of those occasional proteins that travel the wrong direction during electroblotting.
I have used this several times in a teaching lab to get lots of samples blotted with practically no equipment; just the glass plates normally used to cast the SDS-PAGE gels plus saran wrap and rubber bands to hold the plates together.