I am working with 100-120kDa proteins that are expected not to transfer well from SDS-PAGE to a nitrocellulose during semi-dry transfer. However, since our lab doesn't have the wet-transfer cell, I have to get the most of what we have (Bio-Rad Semi-Dry blotter).
After reading various advice on constant amperage vs. constant volts, taking an account for the membrane surface, etc. , I reached to some combination of amperage/time, that gives me "good" transfer. I put the "", because I still have proteins left inside the gel (visualized by commmasie after the transfer), but at the same time I have some molecular weight marker (the one for 150kDa!) that gets onto the filter paper below the membrane.
So has anyone solved the "black box" equation of the semi-dry transfer, using a methodological optimization procedure to determine which are the best settings so that most of the protein of interest is out of the gel, but is on the membrane and is not below the membrane ? Please share.
For me using the molecular weight standard as an indicator of transfer is not relevant, because the total protein amount in 10 microliters of standard is usually about 3-5 micrograms. I was thinking about staining covalently somehow some cheap protein (like BSA) and simply tracking how much die I have left on the gel, on the membrane, and possibly - on a second, back-up membrane. What do you recommend?
Hello. In our experience, transfer of proteins of the MW size you are indicating is not a problem using 10-20 ug/lane, a 7 cm gel size of 1mm, 10 %SDS-PAGE, 13v/30 min, PVDF membrane- For us, this is a common strategy for transfering heavy chain myosin of 250 KDa and the transfering is complete from the gels to the PVDF
I have not done semi-dry method for 12 years after my fellowship training and honestly can not give you a good suggestion. However, I used to stain the gel with Coomassie blue after completion of the transfer. Correct me if anybody knows the real answer, I always have stain on the gel if I let it sit in the solution long enough. The point is the proteins are evenly transferred to the membrane. If the protein is more lipophilic you may try the PVDF instead of nitrocellulose. My junior colleague who was trained in another elite medical school uses semi-dry method using similar setting as Javier mentioned with good results but she uses PVDF. Maybe you should try Ponceau stain to your membrane to show even transfer.
Hi Peter,
I transfer 70kDa to 250 kDa from the same gel of SDS PAGE 6-8% about 10 cm X 8cm using Bio-rad semi dry apparatus. They transfer like blotch! although not always 250kDa but 150kDa which is a dimer surely does.
I perform 20-22V for 1 hour 15 min. Ladder as you mentioned is not an ideal situation coz they are less in conc and what we use is 10 ug generaly per lane so it takes time.
Keep your blots well hydrated, keep your AC on while transferring, and perform it for little longer time. Atleast I have never seen bands left out in my gel.
It works for both PVDF which is always activated using methanol or NC membrane activated by water. I stain the membrane in Ponceau S post transfer to see how well it got transferred.
Hope it works.
Dear freind u have to run 8 % gel and let it seprate well and added 50 ug of protein. Run the transfer for 1.30 hr.
Thanks everybody for your answers! I actually run 40 micrograms of protein in my gel for 2.5 hours to separate well the hypophosphorylated from the hyperphosphorylated form of my protein (Rb), then I run at constant A - 0.003A per sq. cm - 0.13A for 80min and my WB look nice (although as I mentioned there is some protein in the gel and some that trespasses the nitrocellulose).
The point is that I would like to know some straightforward general optimisation procedure for semi-dry transfer when working with new protein. I tried finding such in the manuals of several semi-dry blotters, but I couldn't...
You could have a look at Current Protocols: http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb1008s83/full
You can also try to play around with SDS and MeOH concentrations in your transfer buffer. Normally, up to 0.1% SDS can be used, it will improve protein transfer out of your gel onto the membrane. However, you might loose LMW proteins. I have heard that people use 2 membranes in the sandwich to prevent loss of LMW proteins - if they run through the first membrane, you "catch" them on the second.
Lowering MeOH concentration or omitting it completely is also an option, since MeOH leads to shrinking of gel pores during the blotting, so that larger proteins have more difficulties to migrate out of the gel.
Also, with semi-dry blotting you can consider the use of two different buffers (i.e. discontinous system) on the top and bottom of your sandwich. I have never tried this myself but you should find some protocols online.
Transfer of proteins of the MW size you are indicating is possible using aprox. 10 ug/lane, a 7 cm gel size of 1.5 mm, 11 %SDS-PAGE, 13v/30 min, PVDF membrane. You should test these conditions and compare perhaps with a wet transfer overnight.
Normally I just run it overnight, instead of those two hours that most protocols say. It gets a bit warm, but afterwards you don't see much protein on the gels even with silver staining.
What I think is important here is to know what is your purpose: do you do it to run a Western blot and there is not enough protein to be detected? Or maybe you want to quantify your expression systems and be sure that you had all protein transferred? In either case, I would just load more on the gel than spend hours/days/weeks trying to optimise a protocol that works to some extend.
Kamil,
my objective is - using as little protein as possible and get as stronger signal as possible. This is very important if you have limited quantity of sample (adipose tissue biopsy, for an example) and you need to blot several different proteins.
What happened last week is that I loaded 80 micrograms of protein (from a set of samples for trial) per well, got some nice bands, but then with the real samples I could load only half of it - 40 micrograms. For my surprise, the bands were even nicer- brighter, sharper, better resolved (all reagents and conditions were the same)!
I can speculate that loading more protein actually causes less efficient transfer.
This made me think that it is probable that in western blotting not always "more" is "better". "Less is better" is definitely true for real-time PCR and may be is for WB as well.
What I intended with my topic was not to get a specific recommendation on a specific combination of parameters for semi-dry blotting, but an optimization framework or a decision tree. Many companies have published such optimization decision trees for qPCR and it surprises me I can't find such a thing for semi-dry blotting.
In my opinion, if you are in doubt if your protein of interest is transferring, you have to stain the membranes after 2 hrs of transference with 25 mA, 8-10 V, you can use a ponceau red solution to detect the presence of the protein in the membrane, after the transference...I agree with some previous answers that describe overnight transference protocols, but they have as the worst issue the warming of the equipment in a situation that could be dangerous to the semi-dry apparatus. In my experience a 100 kDa protein must be transfered using a biorad apparatus in the maximal amperage (25 mA) employing something like 10 V as current...Another advice, use PVDF instead of nitrocellulose membranes because they retain the proteins very much more and you don´t wanna see your protein going out to the filter paper.....
Dear Petrov,
There is a manual for general western blotting from Millipore.
And about starndarization, after I have done many myself, I think the protein that you see left on your gel can have 2 causes:
1. You are loading too much protein (what you alredy mentioned as being probable!). The saturation level can be reached and the transport will not take place.
2. Your buffer system is not adecuate for your protein. I have seen that my proteins (130-270 kDa) do not transport well using the one-buffer system. They only get transfered to the membrane using the 3 buffer system: Anode I, Anode II and Kathode.
Additionally, I have seen that many other investigators "equilibrate" their gel shortly in methanol solution. I have stablished that such a step crosslinks part of the big proteins onto the gel and the transfer is incomplete.
I actually have better experience with semidry transfer systems as with wet transfer. I hope this can help you.
Greetings,
Luisa
Hello Luisa,
I got the Millipore guide two weeks ago and I also use a discontinuous buffer system.
What that guide recommends as well is to equilibrate the gel in the cathode buffer for 15 min , which I started to do - their argumentation is that in this way you remove salts and other components from the electrophoresis step and that such treatment helps the gel to reach its final size. It actually makes sense, because anyway the gel is in direct contact with the methanol-containing cathode buffer during the semi-dry transfer.
However, I believe you also make a point for the possible cross-linking!
Do you rinse your gel in ddH2O before putting in over the membrane ?
Yap. and only until I don't see any "bubbles" coming from the SDS. Sometimes in order to remove any bubble between the gel and the PVDF membrane, I dip both shortly in the Anode II buffer and then on the stack as normal.
At the moment I am doing my gels with a system buffer published by Ahn et al (2001) and although teh bands are not HD (high definition), my transfer works EXCELLENT. Good luck.
hi...just a troubleshooting for transferring large proteins....add a pinch of SDS in ur transfer buffer...(~100-200mg/500ml). ...it doesnt affect ur blotting...but helps in effective transfer...
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