*Before you comment with any of the generic troubleshooting advice from the front page of Google, just know I've already tried all that*
No matter how I alter my conditions for wet tank transfer, there is still always A LOT of protein left in my gels. After transfer I always check my membranes with Ponceau S, and there are full lanes of protein on the membrane itself, but coomassie staining of the gel shows a lot if left behind. So my trasnfers are incomplete.
Our lab does a wet tank transfer using the BioRad mini protean boxes with PVDF membranes and Towbins buffer (20% methanol) for 300mA for 1-2hrs (depending on target protein size). So far they all seem to be coming out like the attached picture. I have tried using constant voltage ranging from 60-100V, constant amps ranging from 300-400mA, increasing transfer time for up to 3hrs. I've tried replacing every single buffer and reagent used in the entire western process from gel cast through transfer. I've used no methanol and up to 30% methanol. Tried using different boxes, power units, and electrical outlets. Tried everything from 5% gels to 15% gels and gradients all at different thicknesses (.75-1.5mm), and they all come out more or less the same.
The only thing that helped a little was adding SDS to the Towbins buffer, which still left SOME protein in the gel. But not nearly as much. However that resulted in the lower weight proteins blowing through the membrane. I tried contacting BioRads western blot tech service but all they did was suggest the same generic advice for troubleshooting.
Has anyone experienced this? I am waiting on commercial gels to come to as a last ditch effort to see if some how one of the components used in our gel casting we bad but other than that I'm out of ideas for solving this.
List of steps used or troubleshoots tried:
Tissue/cells homogenized in RIPA
Samples are prepped by boiling in laemelli buffer + BME for 10mins
Gels are cast fresh the day of
Gels and sandwich components are soaked in transfer buffer for 15mins prior to transfer.
Transfer is done on ice with a cooling pack.
PVDF membranes are used and activated with methanol prior to use.
All buffers are made fresh.
10-40ug of protein are typically used per lane and all ponceau staining after transfer shows an OK amount of protein of all weights make it to the membrane, just a lot is left in the gel.
Varying amounts of methanol and SDS have been used in transfer buffer.
Varying transfer conditions and transfer boxes have been tried.
pH of all buffers and even DI water have been checked.
Since PVDF membrane has a maximum limit of how mech protein it can bind/hold, there always has a chance that you get some staining on the gel after membrane transferring.
To maximumly transfer proteins to PVDF membrane, you can use a constant voltage of 50 volts for an overnight Western transfer in a cold room (better with a stirring of transfer buffer during the transfer process to reduce local heat).
Wen Liu
Yes, PVDF does have a maximum capacity (150–160 µg/cm^2), but the 30µg of protein loaded in each lane of the pictured gel should not leave nearly that much behind, if any at all. I have used the exact same conditions in previous labs and have colleagues using the same conditions as well with little to no protein left in the gel after transfer.
Dear Belcher,
I wanted to clarify a few things before discussing the issue.
1. Is there any issue with the experimental outcome due to this untransferred protein in the gel? The answer is No (western blotting only gives relative quantification compared with experiment control).
2. To compare the expression of any decently expressing protein, 10-20µgof protein lysate is sufficient. I can see the tailing of the Coomassie stain in the whole length of the gel loading side, which indicates an overflow of protein lysate into nearby wells due to excessive protein loading, i.e., more than the well capacity.
3. The use of SDS increases the transfer efficiency due to negative charge and linearization of protein. Still, excess use of SDS will lead to increased heating of the transfer buffer, which affects the protein transfer. The gel will stick to the blotting membrane and often require laborious effort in scraping the gel off the membrane (More SDS is recommended only in case of large proteins, which will increase transfer efficiency).
To address the issue, you can try,
4. You can try loading 10-20 µg and use Towbins buffer in a constant voltage of 30 volts for 16 to 18 hours of overnight transfer at 4°C storage temperature to observe maximum transfer.
5. As Wen Liu said, there is a limitation to how much the PVDF membrane can hold (You said 150–160 µg/cm^2 is the maximum capacity, but the protein band in your case is not occupying the whole cm^2. Approx. Ten times less, I guess, i.e., 15-16 µg).
I hope this will help.
@Vinay J.
I'll address your response point by point.
1. YES. There is an issue with a lack of un-transferred protein to the membrane. Especially if you are not anticipating a very large difference between groups. In this case, I am quantifying puromycin incorporation, which requires nearly all of the lane to be adequately transferred in order to properly blot and quantify it.
2. It is extremely common in the life sciences to use WELL above 20µg of lysate. Especially in vivo. As I stated in my question, this problem continues to occur with as little as 10µg of protein. Also there were no overload issues and the surrounding wells/lanes are all free of staining with both Coomassie in the gel and ponceau in the membrane. I chose a random image for reference here that happened to be using 30µg and I only partially washed the gel for a quick picture. Attached in another example where both muscle tissue and C2C12 lysates were transferred using both 10 and 20µg. This is not a problem I've experienced previously even when transferring as much as 60µg, nor it is a problem anyone else I know has encountered despite using the exact same conditions. All of their Coomassie staining shows a complete transfer after 60mins. Mine does not show this even after 120mins.
3. I only tried including SDS to the buffer after encountering this problem and found it did not solve it. I would not call 0.1% SDS excessive (also tried 0.25% and 0.05%).
4. I've tried that. Same outcome. But again, a short high intensity field transfer should be more than enough. Just as it has been in the past. This is a newer problem that just began recently.
5. The rough measurements of my lanes yielded a 0.5cm x 5cm area. So 2.5cm^2. Which in theory should allow for a total of 375µg of protein to bind within that lane (if evenly distributed). I understand SOME specific areas of that lane COULD become over saturated. But for the entire lane to experience this problem does not make sense.
Thank you for your thoughts and time on this issue but I will continue to try and solve this since nothing seems to be fixing it yet. I have since tried commercially purchased gels from BioRad and had the exact same outcome. My search for WTF is going on to cause this continues.
For anyone also having this issue, I solved it after making multiple changes.
1. At some point the wrong PVDF membranes (0.2um) were bought by mistake. After switching back to 0.45um PVDF, the transfers were better, but not complete.
2. Concerned that the methanol may be stripping too much of the SDS from the transfering proteins, causing them to stay in the gel, I lowered the methanol concentration to 10%. This also helped but did not fully transfer everything like I wanted.
3. I then added SDS to the transfer buffer (0.04%) to see if there would be better results with the now correct membranes and that seemed to solve the problem!
So the full solution was using 0.45um PVDF membranes and a Towbins transfer buffer with 10% methanol and 0.04% SDS.
The attached gel is a 1.0mm, 10% acrylamide mini gel that had varying protein concentrations from 10ug-30ug and was transfered for 1hr at 100V.
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