I reuse transfer buffers several times, but I store them in the fridge to prevent methanol for evaporating. Also during transfer it is important to cool down while transferring.

You seem to have loaded very little protein 0.6 µg if I calculated correctly. I usually load between 5 and 20 µg of protein. Which detection method did you use to decide that your proteins didn't get transferred? Is this sensitive enough to detect this small quantity of protein?

It is not advisable to reuse the transfer buffer as methanol may have evaporated/denatured during the transfer that may explain the poor transferring of your proteins to nitrocellulose. Your transfer buffer composition seems fine too.

I reuse transfer buffers several times, but I store them in the fridge to prevent methanol for evaporating. Also during transfer it is important to cool down while transferring.

You seem to have loaded very little protein 0.6 µg if I calculated correctly. I usually load between 5 and 20 µg of protein. Which detection method did you use to decide that your proteins didn't get transferred? Is this sensitive enough to detect this small quantity of protein?

How do u knw that protein is not getting transferred onto the nitrocellulose membrane? If marker is getting transferred onto the nitrocellulose membrane, means that protein is also getting transferred. I think the problem is in antisera or antibody which u r using for the development of blot. .

The first question to ask when a transfer fails is usually if you are sure that you connected the transfer chamber right (do be offended, this has happened to everyone who has done westerns I know). Is your marker transfered to the membrane? If yes, then your protein should be there as well.

Did you try to stain your blots after the transfer with Poinceau Red?

You load very little protein (below 1ug if my quick calculation is right), I usually use between 20 und 30ug for total cell extracts. You might simply below the detection limit of your antibody.

Your buffers look fine and you can of course re-use them without problems for quite a while. The methanol is not going to evaporate if its stored in closed bottles.

It's rather imposobile that you have marker transfered and proteins are not. Maybe the problem is with your protein extracts. You can also play with transfer conditions like concentartion of methanol or the duration of transfer in case your protein is heavili phosphorylated or modificated in the different way and sticks to the gel. Did you try to stain the membrane with Ponceau?

My first question is: how do you know that your transfer doesn't work? Your protein loading seems to be insufficient to be controlled by ponceau red staining on the membrane.

Finally, if you have others detection methods that assume your transfer doesn't work, try to use transfer buffer without methanol that is not absolutely necessary. (I think there was a recent interesting discussion about methanol in transfer buffer a few months ago, on researchgate)

Yes, how do you know that your protein did not get transferred. If it is on the basis of a downstream application such as an immunoblot, then you could have a problem with the blot. This can be easily checked by staining the membrane with Ponceau S, that would briefly stain the transferred proteins pink.

You have also not indicated the amount of protein, in µg terms, that you loaded on the well. Els is right, you need to get the amount correct. Also, at times, not routinely, I have reused the transfer buffer.

I would think about the molecular weight of the protein you are handling. I do not know what support you are using, but if you are using a non-reinforced membrane, then smaller proteins would tend to cross the membrane into the buffer. The 100V 1h run that you gave would work best with proteins in the range of 20-50 kDa. You might have problems in resolving smaller or larger proteins. As a rule, smaller proteins would be transferred faster, while the larger ones slower, so you may need to optimize your transfer conditions.

I think most aspects have been covered in the above posts. Since you are getting your markers, the transfer system/protocol per se seems to be working. A quick sum up of what I would do: 1. check with ponceau to see if proteins have transferred. 2. run a positive control lane of a sample that you know has the protein you are looking for. This should tell you if your antibody is working and the detection system is right. Make sure you load the same amount of protein, though. 3. Load more protein (0.6ug may not be sufficient to detect your protein). 4.do a longer transfer if you are dealing with bigger proteins. 5. you could also try another detection reagent, e.g Supersignal West Dura, or Pico, etc. All the best.

I agree with some of the previous comments, you load a very little amount of protein (0.6 ug). If you want to detect some high abundant protein or an overexpressed protein it can be enough, but for others, I think it is not. You also want to choose the optimal running and transfer conditions according the Mw of your target protein. For bigger proteins it is worth adding some SDS in the transfer buffer (0.025 - 0.5 %). It is important to find the right conc. of SDS and MeOH in transfer buffer. They have opposing roles. SDS helps elution of proteins from the gel, but decrease the binding efficiency to the nitrocellulose membrane. On the other hand, MeOH removes SDS from proteins and improves the binding to nitrocellulose membrane, but can cause the precipitation of some proteins. Anyway, I have the experience that without SDS in the transfer buffer, it is very difficult to blot bigger proteins, let's say above 100-120 kDa. I usually transfer the proteins for 60-90 min with 300 mA constant on ice, with 0.025% SDS in the transfer buffer which is pre-chilled before trasnfer. You also want to choose the optimal total acrylamide % gels according to your target protein. For bigger proteins lower %, for smaller proteins higher % gels. Anyway, the easiest way to test the quality of transfer is Ponceau red staining, quick, cheap and you can reuse the dye several times.

Here are two papers about the re usage of transfer buffer:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685604/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841995/

They are the opposite, so I think you want test it in your own.

You can reuse transfer buffers, but you should store it in the fridge.

Because, the markers have been moved to nitrocellulose then the action of transfer was correct.

You should stain the nitro-cellulose paper with ponceau S red before adding antibody.

Some proteins are transferred difficulty due to the inherent properties.

finally, you should load between 15 and 30 µg of protein.

Agree with the above. While transfer buffer can be reused, it's easily made and should probably be used fresh. Also, what is the overall charge on the protein under these conditions? When it's run on the SDS gel, its charge is masked by the SDS, but when it is transferred, the SDS concentration is different so the natural charge of the protein is more dominant. It may be that it doesn't want to migrate to the nitrocellulose because it has the wrong charge. I agree with previous posts that you should 1)run a positive control 2) run a higher amount of protein 3)post stain the nitrocellulose to rule out any antibody issues.

My experience says it is best not to reuse the transfer buffer, it is relatively inexpensive to make. Keeping everything ice cold is important. Keeping the transfer box in an ice bath in the cold room while you transfer would be ideal. Running your transfer at 100V sounds very high and if you are transferring a small protein you can potentially push it through the nitrocellulose. Do you see small proteins from your markers on the filter? Ponceau S staining is a good idea (depending on your detection method after antibody incubation). Are you confident of your primary and secondary antibodies for detection of your protein?

I recommend transferring the proteins over night using 30 mA at 4ºC. I am pretty sure you will get better results detecting your protein in the NC membrane. In addition, I recommend you not to reuse the transfer buffer! You should to prepare a new one every time you want to run a new transfer.

Good luck.

This article you may find it useful

Electrotransfer of proteins in an environmentally friendly methanol-free transfer buffer

Marco A. Villanueva

http://www.sciencedirect.com/science/article/pii/S0003269707005106

People need to be careful using prestained markers as an indicator of efficient transfer. The dyes that are bound to the protein markers are often ionisable and thus the markers transfer with a much greater efficiency than the other unlabelled proteins. The sensitivity on Ponceau is horrible, so if you are starting with a low amount of protein to start with (an amount that will be easily detected by your antibodies because of signal amplification), you might not see it. An alternative to Ponceau is Direct Blue 71. There is a paper from ~2003 that details the method wonderfully (I forget the author and journal right now). Lastly, have you stained the gel after transfer to see if the protein is still there? This might be misleading though because not all of the protein is transferred because the gel can hold more than the membrane can bind and the remainder stays in the gel.

Thanks all. I will do with high concentration of protein.

I think you should change your protocol. For example increase the time of transfering, or decrease the voltage. Also you should recheck your materials.

I will advice two things only

1) Check the grade of methanol. Extract pure methanol is required, I use Sisco Research laboratory (SRL) methanol.

2) Check the mass of your protein and increase the blotting time if Mr>75 KDa, then, use Tubor blotter by BioRAD and do blotting just for 40 min for Mr>75.

3) if your using other blotting systems, avoid bubbles when building the blotting cassette. I guess it helps

I will advice two things only

1) Check the grade of methanol. Extract pure methanol is required, I use Sisco Research laboratory (SRL) methanol.

2) Check the mass of your protein and increase the blotting time if Mr>75 KDa, then, use Tubor blotter by BioRAD and do blotting just for 40 min for Mr>75.

3) if your using other blotting systems, avoid bubbles when building the blotting cassette.

4) PVDF membrane got high binding power than nitrocellulose. You can change the membrane or consider using PVDF.

I guess it helps

I also need to clarify certain doubts regarding the same question Asiya Parvin addressed.

Which is the ideal voltage and time we can use to transfer 100kDa protein? Should we avoid methanol and methanol from the transfer buffer to get an ideal transfer in this case? Can we reuse the transfer buffer? As the isoelectric point of the protein is 4.7 should we adjust the transfer buffer pH to+2/-2  from this value to prevent any hindrances during transfer .