We Are trying to obtain efficient and reproducible western transfer of a 340 kDa protein. We routinely do westerns for proteins of 140 kDa and below with no problems. Does anybody out there have any suggestions for our 340 kDa protein?
Hello there! I worked for years transferring a 570KDa subunit (darn monster!!!), so here's my 2 cents: mobility (within the gel and outside of it) improved a lot when working with lower total acrylamide gels, as most colleagues have pointed out. 6% would be awesome if it wasn't so difficult to manipulate later... 7.5% was a happy medium for me. But, what worked wonders was the thickness of the gel: try 1.5mm thick gels... big proteins appear to have more room to move there :) I supplemented in the transfer buffer with 0.05% Tween-20 rather than SDS, to use a less chaotropic agent. Finally, I transferred overnight (16 hours) at 20V at 4ºC.
It is of note that it's rather difficult to have a quantitative transfer of such big proteins, but depending on the application, that should not be an issue for interpretation of data, except when you are looking for differences of expression of the protein. If that is the case, let us know!
Best wishes and good luck!
Hii
I have successfully transferred proteins upto 300 kDa with the following modifications:
1) Run your samples on a 6% polyacrylamide gel. Use good amount of protein (30-50 ug per sample)
2) Overnight transfer at 40 V for 16 h or at 70V for 3 h. I personally prefer the transfer protocol for 3h at 70V.
3) Use 0.05% SDS in the transfer buffer (though I found this step optional, but you may give a try with this.)
(1X Transfer Buffer composition for 1 lt: 3.03 g of Tris, 14.4 g of glycine and 200 ml of methanol). We use nitrocellulose membrane in our lab.
Some more info as what buffer you are using and conditions would be helpful. If you are using standard 0.037% SDS and 20% methanol (Sambrook et al.) and a wet blotter, try reduce the voltage (20 - 25 V) and blot overnight.
Semi-dry blotters are not as good for large proteins.
I also made good experiences using the described buffer of Sambrook (48 mM Tris; 39 mM Glycin (pH 9,2); 20% Methanol; 0,0375% SDS). This one worked for up to 270 kDa proteins even using a semidry-blotter and running only 45 min with 15 V and 400 mA. Good luck!
I agree with the previous comments. We work with mucins and usually they need a 6-7% polyacrylamide gel resolving, low voltage and overnight transfer. Check also if nitrocellulose or PVDF are suitable for you protein.
Similar to the suggested above. For 260 kDa, I have used 0.04% SDS, 20% ethanol (to avoid methanol which is very toxic) and 3h at 70V, wet transfer cube. My stock 10X transfer buffer: 58 g/l Tris, 29 g/l Glicine, 3.7 g/l SDS or 0.4% if in solution, Add water for a final volume of 1 L, pH=8.8.
I add the ethanol and water just before the transfer.
Good luck!
In order to achieve reliable transfer of proteins around 250 kDa I follow the suggestions above (wet blot overnight at 100 mA, constant current) and I increase the salt concentration of the transfer buffer two-fold (final 28.8 g Glycine, 6 g Tris per litre). I also add 20%methanol and 0.1% SDS, and run the proteins on a 7-8% PA gel (or 4-12% gradient gel). This works well for nitrocellulose membrane, I haven't tried with PVDF.
Additionally to all the good comments above I suggest to consult papers from Hermann Schägger, who investigated the transfer of very large protein complexes separated by "blue native electrophoresis" from the mitochondrial membrane: eg http://www.ncbi.nlm.nih.gov/pubmed/19882660?report=abstract.
It might be me, but I do not see why you would deviate from any usual western blotting protocol. When you have a suitable gel which is able to resolve your protein correctly (6-7% gels like others have suggested), blotting your gel to your membrane for 1 hour at 100V should do the trick.
We use the Biorad XT MOPS buffer with 20% methanol.
If overheating is an issue, make sure to place your blotting container in an ice-bath at 4 degrees celcius.
If you tried this and it doesn't work; can you describe what went wrong?
If, with all of the suggestions, you are still having problems, go to a large (15x15 cm) gel system, transfer overnight at 4C at low voltage (25), then for 2 hours at high voltage (100).
Hi, there are well established tris-acetate gradient gel systems (3-8%) established as for instance described by Altrock et al, Neuron 37,787- (2003). Such gels are also commercially available (NuPAGE) as well as the required special running buffer and the special blotting buffer (at least no SDS and low or no methanol). Blotting onto PVDF membranes at usual currents for tankblotting (appr. 200 mA), time appr. 2-4 hours (using PVDF membranes proteins do not migrate through but stick to the membranes thanks to no SDS in the blotting buffer) One of the proteins to be detected was bassoon which is of the size you are interested in.
It's not necessary to add methanol to your transfer buffer if you're using pdvf (you need it to activate your membrane with methanol, though). Methanol makes it harder for proteins to come out of the gel. If you don't add methanol, you have to soak your gel in transfer buffer for a while. I usually take the middle road and add 50ml methanol per liter of transfer buffer.
In our lab we also make our gels with a 80:1 ratio of Acrylamide:Bis-acrylamide. Therefore we buy our acrylamide and bisacrylamide seperately. This makes our gels more stretchy, so cut your blots somewhat bigger than your gel. This ratio is supposed to get a better seperation and your proteins will come out of the gel more easily.
Good luck!
Hello there! I worked for years transferring a 570KDa subunit (darn monster!!!), so here's my 2 cents: mobility (within the gel and outside of it) improved a lot when working with lower total acrylamide gels, as most colleagues have pointed out. 6% would be awesome if it wasn't so difficult to manipulate later... 7.5% was a happy medium for me. But, what worked wonders was the thickness of the gel: try 1.5mm thick gels... big proteins appear to have more room to move there :) I supplemented in the transfer buffer with 0.05% Tween-20 rather than SDS, to use a less chaotropic agent. Finally, I transferred overnight (16 hours) at 20V at 4ºC.
It is of note that it's rather difficult to have a quantitative transfer of such big proteins, but depending on the application, that should not be an issue for interpretation of data, except when you are looking for differences of expression of the protein. If that is the case, let us know!
Best wishes and good luck!
run your protein in 6% gel. ensure you use high molecular weight marker. do wet transfer at 400mA for 4 hrs. at 4degrees. it will transfer the protein as high as 500kDa
We have just completed an investigation into this very problem. Our protein is >250kDa. For the best transfer we used PVDF membrane with transfer buffer made up with only Tris (5.8g/L) and glycine 2g/L in distilled water (NO SDS or methanol). SDS although can help with solubilisation of proteins also can interfere with transfer, methanol is not necessary for transfer to PVDF (only for pre-wetting of membrane) and can also dehydrate your gel and remove SDS from proteins leading to poor transfer. You may also want to cool as you transfer to prevent overheating (cool block or ice box or 4C). Obviously you need to have a low enough acrylamide concentration 8% or less but I guess you know that. Transfer was at 100v for ~1hr using the wet transfer technique (semi dry may lead to poor transfer of larger proteins)
Look Biorad website/abcam they both have good discussion/protocols to get you started. We did try a number of different protocols +/- SDS/methanol and the above was the best.
good luck
Hi, I have obtained better results to transfer 450 KD (and higher) proteins from BN-PAGE using the next condition: transfer buffer: 39 mM glycine, 48 mM Tris-base, 0.037% SDS, pH 8.3 for 4 h at 100 V constant voltage, without methanol. We tested different methanol concentrations in the transfer buffer and the best results were obtained without methanol.
You may do an over night transfer in cold room using about 40 V . This has worked for me when I tried to blot MUC-1 which is also a high molecular weight one. Make sure your stirrer is also working and can work in cold conditions.
I had very good experiences by using self-made Tris-acetate gels (around 6% AA) and a Bicine/Bis-Tris containing transfer-buffer. As we are working here in our lab with huntingtin (~350 kDa) and previously having problems transferring or detecting it, we started using precast and selfmade TA gels and the respective buffers. We perform the transfer at 200 mA for 2 h using a PVDF membrane. Good luck!
Transferring at a lower voltage overnight in the cold room will help the transfer of high MW proteins, reducing your gel to 8% and running the gel till just the higher MW markers are visible on the gel will also help
Increase SDS and lower methanol percentage combined with ON transfer "lower voltage".
I had good result for the detecction of a ~330kDa protein by using 4-12% NuPAGE gel and the iBlot transfer system (nitrocellulose transfer stack, program P3, 7:00 min).
Hi,
Something I had an issue with was the solubility of my protein when doing high molecular weight proteins. Apart from the solutions already suggested I would say ensure that your samples are well boiled before you load them on to the gel. Also give them a good vortex. That combined with increasing SDS and doing overnight transfers did the trick for me.
Good luck!
Hi,
We worked on approximately same KDa recently. We use Abcam protocol. It's so important to use 6% gel and for transferring put your membrane in 200mA for 4h. For efficient transferring carry it out at 4 degree or put your system in ice.
I have also tried the BN page Conditions as described by Gabriel Valdivieso above to answer your question. It worked without methanol. I was working on OXPHOS Complex-1, which is about 1Mega Dalton in Size. You can see the BN PAGE protocol in more detail going through the link below:
http://www.nature.com/nprot/journal/v1/n1/abs/nprot.2006.62.html
All the Best
Manoj
I've had great success measuring denatured ATM (350kDa) and native TSP1 (450kDa). I use precast gels (4-15% Criterion TGX) and transfer using standard Tris/Glycine buffer (0.5X + 10% methanol) onto nitrocellulose. Initially, I had a lot of problems with the transfer and tried several apparatuses. What worked best was the simplest - Genie Blotters (I think I bought it from RPIcorp). I typically transfer the gels for 30 minutes (24V) at RT. For TSP1, I transferred for an hour.
We routinely perform western blots of proteins from 25 up to 550 kDa, and we are able to detect them on the same membrane. Our conditions are:
- Gradient precast minigels TGX 4-20% (from Bio-Rad).
- Standard SDS-PAGE (tris-glycine buffer)
- We use the mini-Protean system for electrophoresis and wet transfer. In general, I do not recommend semi-dry transfer for proteins larger than 80-100 kDa.
- We transfer onto nitrocellulose for 75-90 minutes at 200 mA, or 100V. We use a cooler and gentle mixing to prevent overheating.
- We always perform a Ponceau staining to check transfer efficiency, and we also load a prestained protein standard (Kaleidoscope).
Best of luck!
Precasted gradjent minigel usually works nicely if you want to separate a wide range of proteins. If your scope is to separate 340kDa proteins than a minigel with low polyacrilamide % (4% or 6%) will work perfectly.
The gradient gels will allow the separation of reference proteins if you want to use them as marker of equal load of each lane; ponceau will work for the simple gels.
Transfer O/N for big protein is usually preferred at least with wet transfer systems.
Good luck!
Hi,
You should try the following conditions:
- 6 – 7 % Polyacrylamide Gels
- Reduce the quantity of methanol in your transfer buffer (if you use normaly 20%, reduce to 10% or less)
- Add a small amount of SDS to the transfer buffer (up to 0,1%)
- Transfer overnight at constant voltage (40V) in a refrigerated room.
Hope it help,
To all of you lovely scientists out there who posted extremely helpful answers to my question- a heartfelt thank you! We considered the answers and came up with a 'consensus', involving 10% methanol, .05% SDS and the NuPage antioxidant, 1.5 h transfer at 125 mA. The procedure worked perfectly first time and we are delighted! Transfer of the 340 kD protein worked well, even from a 12 % NuPage gel! This is my last project before I retire to follow a new career as a jazz and latin pianist- so it's great to finish on a high. Thanks again to all of you from the four corners of the world and the very best of luck in your own careers! Ranmohan Wickremasinghe
Dear Ranmohan,
can you please tell me what membrane did you use ?
thanks
my protein of interest ~ 450kDa. I use usually 6% resolving and 5% stacking gel. I run gel for 4-5 hr/200V. Transfer 20% methanol/90min.
Some time I got and some None. what the best to do???????
You can try transfer with no alcohol and 0.5ml of 20% SDS; 300v for 4 hours (wet tank transfer with ice packs) or overnight wet tank transfer in cold room with 30v.
AS for gel, you may also want to try using gradient gel. BUt i think you should first try with wet tank transfer overnight.
Please also add information about the membranes (PVDF or nitrocellulose).
Thank you in advance
If you transfer with a shorter protocol of around 3 hours, is this at room temperature, or in a cold room? If at room temperature, to you use pre-cooled transfer buffer or keep the tank on ice?
Thanks!
I used to work with high molecular wight proteins such as BRCA2 and ATM in 6% Polyacrylamide Gels and transfer for 16 hours at 4c all were very good.
Hey Olivia. When transferring a HMW protein, many factors should be taken into account, like the how hydrophobic of the protein and its charge. One has to try various methods until finding the optimal one for your specific protein of interest.
the most traditional way is of course to use a wet tank blot and perform a very long transfer. Some companies offer special gels and transfer buffers for HMW proteins specifically, for example it is recommended to avoid methanol completely in the transfer buffer since large proteins might precipitate in it.
Here are some useful tips you may consider in your optimization procedure:
https://bitesizebio.com/24228/five-fast-tips-for-blotting-large-proteins/
Best of luck!
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