@Cecilia

Increasing the methanol concentration will generally reduce the current during the transfer if you use constant voltage and cause it to run slower and cooler. If you use constant Amps, it will increase the voltage and cause it to run hotter and possibly faster. Methanol's dielectric constant is much lower than the transfer buffer's dielectric constant. Adding more methanol is reduce the solution's dielectric constant and increase the buffer's resistance and reduce it's conductivity. The consequence of this is that it will take longer to transfer under constant voltage conditions compared to 20 or 10% methanol, but the binding will be stronger to the membrane. The way to get around the increase time to transfer is to use constant power (watts) to transfer your protein, which will allow a higher voltage to compensate for the increased resistance, and decreased current. Many powersupply units don't have an option for constant power so you may have to just live with transferring it for a longer time. You can also run it at constant amps, but you may need to reduce the current than when you do 20% methanol, as it may overheat. Since the resistance is higher at 30% methanol, in order to push the same amount of current the voltage needs to increase, which leads to increased total power, hence the higher heat output.

If you increase the methanol concentration waaaay high like 80%+ you can melt the nitrocellulose. This happened when one of the techs mistook nitrocellulose for PVDF, when they were trying to "wet" the PVDF membrane. nitrocellulose melts in 100% methanol haha.

I never use methanol in my transfer buffer - ethanol works just fine and is much safer to use (methanol is toxic...). I use the Transfer Buffer from Nupage (Life Technologies), but in denaturing conditions. It may be different for native PAGE...

http://www.carlroth.com/website/en-nl/pdf/Transferpuffer_E.pdf page 4

This link may useful for you.

Http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152___15602542_-1

Here's some advice from the Thermo Scientific site, where they suggest omitting methanol to maintain the activity of enzymes. Some info for semi-dry methanol-free transfer is available at the end of the document:

http://www.piercenet.com/browse.cfm?fldID=C946AFF6-CCD2-A65C-8A96-03C4AC3E1E07

Good luck!

Hi, methanol fixes your protein and band does not diffuse. you have to use some fixing agent to keep your protein bands crisp. methanol (10-20%) doesn't affect protein conformation. Anyways after SDS-PAGE, your protein is denatured. For native PAGE, protein size will be different and you definitely need a fixing agent again. You can do dot-blots or ELISA based techniques instead of western if your protein conformation is that important.

You can replace Methanol by Ethanol with no modification of the quality of your experiment.

Replace methanol with Ethanol.

Hi Nishant,

Of course you can transfer without methanol, in any kind of transfer (continuous, Two buffers type of three buffers type). But in some cases, dependently of the type of gel and acrylamide concentration, you will need to fix your protein..in this case replace methanol with ethanol. Secondly, as Carla said earlier, it depends one the membrane type. If you have to use PVDF, you cannot avoid methanol

Hi Nishant,

In fact even if you have a PVDF you can use ethanol for pre-wetting and in the transfer buffer. We do it in our lab. For reference you can look through "Western Blotting Using Polyvinylidene Difluoride (PVDF) Membranes" by Invitrogen Life technologies (according to them you can even use the isopropanol) and a manual for Amersham PVDF HyBond-P PVDF Membrane note 3.1. We usually pre-wet it for a minute or so in pure ethanol then wash and go on with equilibration in transfer buffer.

Here is also some facts from www.agrisera.com WB recommendations:

The presence of alcohol in the transfer buffer will decrease protein mobility out of the gel. It will also reduce pore size of the gel, while it will improve binding to nitrocellulose as it removes SDS from proteins and increases their hydrophobicity. Higher molecular weight proteins might not be transferred completely if methanol is present in the transfer buffer. Change membrane to nitrocellulose, omit methanol from transfer buffer, while adding SDS and increasing field strength.

Methanol is only necessary when nitrocellulose is used. If using PVDF, methanol can be removed from the transfer buffer altogether, and you just need to activate the PVDF with methanol before assembling the gel/membrane "sandwich" according to the right order.

First, PVDF is not nitrocellulose! You cannot change just like that. Of course you can change methanol with ethanol, but be it will change the hydrophobicity of the membrane. Finally, be carefull with SDS, it change field stregnth but also highly modify "binding" of proteins to the membrane...check every point .

For me, fisrt, try nitrocellulose without methanol for transfert your native gels

Hi Nishant

Both Ethanol and methanol are used in western blotting for fixing and both fixes protein by reducing the solubility of protein molecules and by disrupting the hydrophobic interactions that give many proteins their tertiary structure. If your protein is of higher molecular weight then you can totally eliminate the methanol from transfer buffer and reduce the transfer time.

Yes you can use ethanol or methanol instead of the methanol

http://www.ncbi.nlm.nih.gov/pubmed/17850757

I remember a very nice article comparing different alcohol / SDS / re-use of the transfer buffer, but impossible to find it again

The purpose of using Methanol in transfer buffer it to remove SDS and to increase the hydrophobicity of the proteins which will allow it to transfer well into the membrane. Higher methanol percentages will affect transfer of higher molecular weight proteins because in addition to removing the SDS from gel and increasing protein hydrophobicity, methanol also decreases pore sizes. While this is helpful in some cases, it is not always helpful. In the absence of methanol though the proteins will swell and will cause band distortion. I have never replaced ethanol with methanol so I cannot comment on that except to say that there are many papers on how ethanol can be used to replace methanol. I have used lower concentration of methanol with good results.

yes you can use ethanol instead of methanol. I used to do this. work very well.

The most important step where methanol (or ethanol) is important in wetting the membrane complete. So you need only small amounts of alcohol ... and you avoid any left (small) air bubbles in the membrane which can disturb later on the transfer.

Thank you all for the valuable suggestion.I transferrred the protein without methanol using PVDF and it worked. The reason I wanted to avoid methanol in the transfer buffer is to know whether methanol can affect the protein conformation. I want to know if a protein in the monomeric form can chages to oligomers in the presence of methanol ( I detect with oligomer specif antibody) But even after elimination of methanol i can see that oligomer specific antibody reacts with the monomers (suggesting a change in the monomer conformation?).

You can use dry-blot apparatus. It will remove all hazards of using buffers. I use it and getting very good results.

Dear Nishant

We usually use this blot buffer (48mM Tris, 39mM Glycin, pH 9.2, 20% Methanol).

The role of methanol in this buffer is to help good transfer of small proteins to membrane and also to prevent swelling from heating and to keep proteins adsorbed to membrane. Also there are some protocols for blot buffers without methanol; but transfer will not perform optimally.

Hi everyone,

Is concentration of Methanol will affect the transfer process? If affects, how?

What is the optimum percentage of methanol used as a part of transfer buffer?

Is there any concentration difference of methanol while using with nitrocellulose and PVDF membrane or same concentration applicable for both membranes? if is there difference in concentration, what should be that difference?

Dear Ravinder

We used similar concentration of methanol (20%) for both nitrocellulose or PVDF membrane in the transfer buffer and find good results.

If you want to to remove the bound Coomassie blue after transfer, you should to soak membranes for 10 to 30 min in 45% methanol (nitrocellulose) or 100% methanol (nylon or PVDF). please pay attention that this step is not needed if using chemiluminescent reactions or radiolabeled protein A for immunodevelopment.

If you have any question please dont hesitate to contact me.

Hi Masood

Thank you, but we are using 10 % of methanol in my lab. Is not sufficient to get good results or effective transfer. If not please can you explain it.

Dear Ravinder

The role of methanol in transfer buffer is to help good transfer of small proteins to membrane. Also, methanol strips the SDS off of the proteins, which changes the charge of the proteins and causes the gel to shrink. Without methanol or using lower concentration such as 10% transfer of protein will not perform optimally.

For many protein transfer applications, particularly applications involving high molecular weight proteins, it is possible to eliminate methanol from the conducting solution altogether. When working with larger, high molecular weight proteins, elimination of the methanol results in a significant increase in protein transfer efficiency (For example, Thermo Scientific Co. produce 10X MeOH-free buffer for use with wet-tank transfer apparatus).

Hi Masood,

Thank you for your valuable suggestions. Finally what your saying that 20 % concentration is optimum and in case of higher molecular wt proteins we will get good transfer if we eliminate methanol. is it the one?

Dear Ravinder

if you use methanol, the transfer of small protein will be better (simultanoeusly, high MW protein will be transfered with or eithout methanol). Please read the following article:

- Gallagher et al. (2006) Immunoblotting and Immunodetection. Current Protocols in Molecular Biology 10.8.1-10.8.28. DOI: 10.1002/0471142727.mb1008s83.

Summary:

Large proteins:

More SDS (up to 0.1%) to avoid protein precipitation / less methanol (10% or less, in case of PVDF possibly absent in the buffer at all) to avoid SDS removing and to enhance gel swelling

Small proteins:

Less SDS (or absent for really small proteins) to enhance binding to membrane, methanol kept at 20%

Have you ever used 30% methanol transfer buffer to improve low-molecular weight (14KDa) transfer? What is the risk of increasing methanol too much?

@Cecilia

Increasing the methanol concentration will generally reduce the current during the transfer if you use constant voltage and cause it to run slower and cooler. If you use constant Amps, it will increase the voltage and cause it to run hotter and possibly faster. Methanol's dielectric constant is much lower than the transfer buffer's dielectric constant. Adding more methanol is reduce the solution's dielectric constant and increase the buffer's resistance and reduce it's conductivity. The consequence of this is that it will take longer to transfer under constant voltage conditions compared to 20 or 10% methanol, but the binding will be stronger to the membrane. The way to get around the increase time to transfer is to use constant power (watts) to transfer your protein, which will allow a higher voltage to compensate for the increased resistance, and decreased current. Many powersupply units don't have an option for constant power so you may have to just live with transferring it for a longer time. You can also run it at constant amps, but you may need to reduce the current than when you do 20% methanol, as it may overheat. Since the resistance is higher at 30% methanol, in order to push the same amount of current the voltage needs to increase, which leads to increased total power, hence the higher heat output.

If you increase the methanol concentration waaaay high like 80%+ you can melt the nitrocellulose. This happened when one of the techs mistook nitrocellulose for PVDF, when they were trying to "wet" the PVDF membrane. nitrocellulose melts in 100% methanol haha.

Yes you can transfer your protein to membrane without using methanol but make sure that your protein of interest should of high molecular weight. Methanol usually help in transferring small molecular weight of protein. Even if you have less quantity of protein methanol enhance the transfer of protein on membrane. Good luck

hi all,

if we use 20% methanol, what would be the optimum transfer condition? It was suggested by Life Technologies to perform transfer for 90 mins at constant 200 mA but this condition was only for 7.5% methanol - containing transfer buffer. Thank you in advance. 

Dear Researchers

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.

1. May i only reduce the methanol to 10% or 5% and omit SDS keeping the transfer 100v/3 hrs to transfer both.

2. using 20% methanol and 0.01% SDS in transfer buffer but reducing the time to 1hour.

3. using 20% methanol and 0.01% SDS in transfer buffer but using 0.22 pore size membrane instead of 0.45???

kindly help it is really important

Hi all,

Want to share my experience as well.

Protein transferred: ~60kD or less.

Transfer Buffer: Without Methanol but pre-activated PVDF membrane in it.

Result: Bands obtained with/without methanol in transfer buffer are identical.

Note: Should be taken care while transferring larger proteins.

Thank you so much dear Dr(s) for your valuable answers appreciate it