Proteins either bind well to nitrocellulose, and then they don't come off, or they bind not so well and come off again and the latter will cause problems regardless of how you do it. Proteins can also saturate the binding sites on the nitrocellulose, and when that happens any surplus goes through and may be detected on a second sheet you put behind the first, but there is really no point in doing so because it won't change anything. Saturation can happen whenever you have a really strong band (visible on the gel by Coomassie stain), and when the protein of interest runs at the same molecular weight as the strong band, it can even be prevented from binding by competition. This can be overcome by changing the percentage of the gel, loading slightly less protein and running the gel slower and longer for better separation prior to blotting. The main issue you should consider whilst transferring a large protein is the progressive removal of reducing agents and SDS during the transfer. This can lead to partial renaturation of the protein and epitope masking. Sometimes re-folding may cause issues with transmembrane domains, leading to aggregation and poor transfer. So in other words, if you do a transfer slowly overnight, the protein may have a different conformation on the nitrocellulose than when you do it quickly in 1-2 hours. I generally like to carry out a Ponceau stain prior to blocking and subsequent antibody incubation, because the 10% acetic acid can re-establish denaturation and exposure of antigens recognised by a polyclonal antiserum. After making a photograph and destaining the Ponceau with PBS-tween, I usually dry the nitrocellulose by blotting on whatman 3mm paper, to be kept until I am ready to do the actual western blot. When the nitrocellulose is dry, no protein will renature, and I am free to decide when and how I will do the western blot.

I like to run my gels and blot them during day-time. I usually like to do the primary antibody for longer times, so I like starting a western blot in the morning so I can give the primary antibpody maximum time to home in on the antigens. In contrast, I like to incubate secondary antibodies for just 30-45 minutes because they are usually present in excess. Working with large proteins is trial and error, so protein work never gets boring. :)

This can indeed happen, but its mostly a problem for small proteins, if I remember correct membrane blow-through is mostly observed for proteins

I've seen going through membranes proteins of sizes up to 70 kDa when using semi-dry blot. However, with wet blotting I've never seen it even for the small proteins (of course you must use lower current *for overnight blotting).

EDIT added "*for overnight bloting" to make it clear

It is really possible to 'over transfer' proteins. You have to try different transfer times and voltages to make sure that you don't 'over transfer' your proteins. As control you can use prestained protein markers. If your marker appears on the backside of your membrane you have 'over transfer'. Good luck!

Personally I've never encountered 'over-transferring' proteins through a membrane. I've only ever used PVDF membrane so I'm not sure if that is a factor (plus I've only done it for 5 years). It was common talk in a lab I used to work in that adding 1% (w/v) SDS to the transfer buffer for semi-dry transfer helped the transfer of high molecular weight protein (>100 kDa). I never tried it personally, but it might be something to read into.

For a large protein like you're talking about, I ALWAYS did "low and slow": wet-transfer, in the cold room, overnight, low voltage. This was for RyR2, which is pretty huge considering (~550 kD). There are a LOT of factors that go into a successful HMW blot. I recommend looking at Invitrogen's protocols for HMW Westerns...they were really useful. Gives good ladders, gradient gels, and whatnot for differentiating HMW proteins.

Oh, and I always used nitrocellulose membranes for this. Just in case it's relevant.

I never tried it myself but I heard about the possibility to put a second membrane "after" the first one, so if your protein goes through the first one, it will be caught be the second.

And for the rest I would agree to the others: Do it slow, have a look at pre-stained markers blotting through and try different conditions.

If you dont want to shift to wet transfer then, use of a discontinuous buffer system (2 pH- 2 buffer) could prevent / reduce ''overtransfer'' and help in transferring large proteins better.

How do you assess over transfer of proteins into a membrane?

Be careful by the addition of SDS. To much SDS will impede protein binding to the membrane.

PVDF reduces the problem of bleed-through due to the smaller pore size compared with nitrocellulose. Furthermore, PVDF comes in at least two pore sizes, 0.45 (Millipore Immobilon-P, Immobilon-FL) and 0.2 micrometers (Immobilon-PSQ). Histones below 20 kDa will transfer right through Immobilon-P but not through Immobilon-PSQ, for example.

Related topic - small proteins come off the gel much more quickly but also to a greater degree than larger ones.

Yes you can get "over transfer" especially with smaller protein or larger ones with too long transfer/high current transfers. Somel people add 2 membranes together to catch this. I have never done this. But another potential problem with 'over transfer' is that proteins can get modified and can lead to epitope damage, so antibodies may not recognize it as well so reduced western signal.

I have always used TGX gels from BioRad and transferred the proteins at 30 V overnight in the cold room with Tris/Glycine/Methanol buffer. It is very unlikely to have bleed-throughs with such a high molecular weight protein regardless of the pore size of your membrane. The most common problem encountered in such circumstances is failure of the proteins to migrate from the gel. This can however be improved by adding a little SDS to the transfer buffer. Hope that helps.

I did a WB once for a 40 kD protein and certainly had a bleed through on NC-blotting paper (Biorad submarine Glycine/Tris). PVDF gave better results (Glycine/Tris/Methanol), but shorter transfer time helped me then too. For small proteins I rather use the smaller pore PVDF membrane (0.22um). PVDF has a more stable negative charge and is hydrofobic, this usually results in a better signal to noise ratio.

Proteins either bind well to nitrocellulose, and then they don't come off, or they bind not so well and come off again and the latter will cause problems regardless of how you do it. Proteins can also saturate the binding sites on the nitrocellulose, and when that happens any surplus goes through and may be detected on a second sheet you put behind the first, but there is really no point in doing so because it won't change anything. Saturation can happen whenever you have a really strong band (visible on the gel by Coomassie stain), and when the protein of interest runs at the same molecular weight as the strong band, it can even be prevented from binding by competition. This can be overcome by changing the percentage of the gel, loading slightly less protein and running the gel slower and longer for better separation prior to blotting. The main issue you should consider whilst transferring a large protein is the progressive removal of reducing agents and SDS during the transfer. This can lead to partial renaturation of the protein and epitope masking. Sometimes re-folding may cause issues with transmembrane domains, leading to aggregation and poor transfer. So in other words, if you do a transfer slowly overnight, the protein may have a different conformation on the nitrocellulose than when you do it quickly in 1-2 hours. I generally like to carry out a Ponceau stain prior to blocking and subsequent antibody incubation, because the 10% acetic acid can re-establish denaturation and exposure of antigens recognised by a polyclonal antiserum. After making a photograph and destaining the Ponceau with PBS-tween, I usually dry the nitrocellulose by blotting on whatman 3mm paper, to be kept until I am ready to do the actual western blot. When the nitrocellulose is dry, no protein will renature, and I am free to decide when and how I will do the western blot.

I like to run my gels and blot them during day-time. I usually like to do the primary antibody for longer times, so I like starting a western blot in the morning so I can give the primary antibpody maximum time to home in on the antigens. In contrast, I like to incubate secondary antibodies for just 30-45 minutes because they are usually present in excess. Working with large proteins is trial and error, so protein work never gets boring. :)

You can run pre-stained protein ladder within your gel to validate transferring efficiency of your proteins with naked eye after blotting.

Just try to validate by running a western overnight for both a larger and a smaller housekeeping protein and let us know!

Thank you for all your responses!

We would always do a transfer overnight and I've never had a problem detecting proteins ranging from 40-120kDa (that's what I was working on).

oh, and we use PVDF membrane

We are using the semi dry system of bio-rad company. It takes only 20 min to transfer proteins (around 100kDa) on nitrocellulose membrane at 20V. We can over transfer the proteins by simply increasing the transfer time. When we increase the time period protein will pass out from the membrane.

http://www.sciencedirect.com/science/article/pii/S0003269797920613

Yes, of courseyou can over transfer the proteins. The protein binding to membranes is not covalent, so if you transfer too long the proteins will shoot out the other side. Using PVDF helps in longer retention. Also, if you can't optimize the transfer time, you may put 2 or 3 layers of the membrane so you can catch the protein on one of them! Generally larger protein transfer slower. Farhad

Yes it is possible to overtransfer the proteins depending on the method you use. Personally, I use the semi dry discontineous blotting method,tranfer time is 2hours at 50mA for one gel (using Amersham Biosciences Uk) and I have always had a good transfer.You can try it

If you have a 'large' protein it is unlikely to transfer all the way through a 0.45um PVDF membrane; I tend to run larger protein >120kDa on 6% gels and then still transfer for 1h at 100V- and I achieve great transfer from the lower % gel (coomassie the gel post-transfer to visualise proteins 'left behind' on gel).

I have heard of another lab that transfers o/n at 4oC running at 30V -so you could try that also.

Yes it is possible to do over transfer of proteins in Western Blotiing experiments, but then do not try to pull out all the hairs from your head if it drives you mad with the results.

when trying to blot my 150 kDa and actin 42 kDa by tris/gly/15% methanol, 100 V for 4 hours the smaller one blotted great in nitrocellulose whereas only small amounts of my large protein blotted.

But when i added 0.01% SDS to the transfer buffer 100V/3 hours, great large protein blotting whereas mostly all my actin blow over as i stained the gel after blotting and no proteins were detected in all lanes.

what do you suggest to blot both. kindly help me.