Cutting the western gel into two parts; one for the high molecular weight proteins and one for the smaller ones. This would allow optimizing transfer conditions for more than one target protein.
Would you recommend or discourage this?
It works, as I have done it before. We had one protein that was better on PVDF and one that was nitrocellulose, so I cut the gel and did separate transfers. Make sure you use a sharp tool like a scalpal or razor blade to the gel doesnt tear. It will be good to use a molecular weight marker on each side as well so you know where to cut across the gel.
One issue is your loading control. Because they will potentially now be on two different types of membranes, you will not be able to strip and re-probe for a loading control on the same membrane. This is not a huge issue as many times people cut the membranes anyway to probe for the loading control. Just keep it in mind.
Good luck,
~Adam
It works, as I have done it before. We had one protein that was better on PVDF and one that was nitrocellulose, so I cut the gel and did separate transfers. Make sure you use a sharp tool like a scalpal or razor blade to the gel doesnt tear. It will be good to use a molecular weight marker on each side as well so you know where to cut across the gel.
One issue is your loading control. Because they will potentially now be on two different types of membranes, you will not be able to strip and re-probe for a loading control on the same membrane. This is not a huge issue as many times people cut the membranes anyway to probe for the loading control. Just keep it in mind.
Good luck,
~Adam
It is fine to cut a gel before transfer but you might want to run 2 weight markers on either side of the gel so you can get a straight line through the weight you want. It is fiddly though.
Great. Thanks colleges for your tips.
Jai, please let us know if you see something we missed.
I had posted an answer but did not read properly, I advise to cut the membrane, not the gel! I do it often for 3 proteins at the same time, for example at 80, 60 and 30kDa (loading control). You just have to know your MW marker (and your ponceau if you use) and where your band stays.
At least in precast gradient gels its very reproducible and easy to do (ruler and scalpel advised)
I also agree with Daniel. I have also done in similar way and it is very reproducible with precast gels.
Fantastic. So you do a 3-day job in one day, Daniel. But a 14 kDa protein, added to your list, will need a special transfer conditions. I think it will be even faster to do both cutting the gels then cutting the membranes ;) And you are right Jai, I have to use a good wight ladder to know where I am cutting.
Typically one would cut the membrane after transfer as transfer conditions do not depend too much on the MW of the protein. Of course for this to work you would have to add maker proteins so you would know where to cut the blot. Another option is to make a small cut in the blot corresponding to the size of the protein of interest. In the past I have carried out numerous transfers and western blots for visualizing alpha-tubulin (55 kDa loading control) and g-H2AX (14 kDa protein of interest). I always cut my blot after the transfer at the 33 kDa marker. I never had any problems transferring small MW proteins as I used PVDF and 10% SDS in the transfer buffer.
That's a good point Haitham. As I mentioned we use precast 4-12% gradient gels and fast dry blotting through the iBlot apparatus. In this system very often you manage to transfer proteins from 10-150kDa using the same conditions and having sufficient efficiency to see them all. Obviously it depends on the abundance and specific characteristics of each protein you are interested, the type of membrane porosity and the transfer system you use.
I would discourage this behaviour of cutting gels in half the across the size gradient. Run two gels. Transfer under recommended conditions for target protein types. The time you spend fiddling and messing around with cutting gels is not worth it. Here's some advice from someone who spent earlier research years doing a lot of this optimization stuff you refer to. Optimization is something which should be done only when required for a project such as for example improving expression of a target protein. The goal of the Western is just to detect your protein by molecular weight. Westerns are not very quantitative so optimizing the transfer is not useful unless one is losing all detection. Repeatable data is more important than "pretty" data. Get it published and move to the next thing.
How about using PVDF then cut the membrane after transfer. I have never expeienced a protein will stay on nitrocellulose better than PVDF but I guess anything is possible. However, the pore size of PVDF is 0.2 um so both large and small proteins will stay.
I got some great tips here. Thanks all for wonderful help. Jeff, your "Get it published and move to the next" is a huge one! It should be printed with Gold first thing on the lab door :)
Generally, you can maximize the transfer of your target protein by playing with the methanol % in your transfer buffer. Methanol deter the transfer of large MW protein because it strips the SDS off. In general 10% methanol is good practice. Depending on the protein you are looking at, you can increase or decrease methanol % to suit your own need. You can also stain your gel after transfer with Coomassie blue to see how efficient is your transfer of proteins of different MW. Cutting the gel before transfer is not really a great solution as you are after all only dividing your protein group to two weight categories, the effect on proteins which size are close to the border is still not gonna be hugely different. Alternatively, you can use pre-cast gradient gel as those things can range from 4-15% or 20% on the same gel and will allow you to see everything and transfer is generally very efficient on all sizes proteins.
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