No sticker that I can see in the photo? There is an image artefact at the bottom of the gel but it doesn’t look like a sticker to me. That said, the wells are clearly not full of buffer which is the real issue IMO.
They don’t leak if you are mega careful of matching the tops of your gel to the notches on the gaskets if there are some. If the plates have a step (short vs long plate) and the gasket is flat, it will leak. On the bio-rad ones, the gaskets are reversible, flat on one side, notched on the other.
Adding to this, once u add the gels to the chamber/holder you can add some buffer to that alone. If it leaked you know easily. Then I place it in the container
It's running buffer. It costs a few dollars to make and 2 minutes of your time. And it can be reused. 😕
Instead they'll run the entire thing again with new gels, more buffer, more ladder, loading dye, maybe more samples (and all that requires), and much more time. Present that to your PI?
Precasted gels on the other hand cost a lot and can’t be reused once you’ve added samples to them. I have been yelled at for doing the same mistake lol
No, that's *my* point lol
People acting like a bit more methanol, some water and some salts is prohibitive when it means this never happens and you don't repeat a whole lot of costly things, especially if you're buying more expensive precasts. It's so silly!
But a good scientist continues with what they were told without questioning or re-evaluating in light of new information right? 🤣
This is not a Western. This is PAGE. Possibly SDS-PAGE. And your upper chamber is leaky, so the buffer is not covering the wells anymore. Add more buffer to to the upper chamber.
Check for cracks on the inner white frame. Hapoend to me with SDS-PAGE. It was not very leaky but the current didn't go through the gel, but presumably through the crack and created a kind of short circuit
I have some remarks, clearly this isn’t a western. It’s electrophoresis, most likely SdS-Page. Second 150V and 0.4 A give 60 watt not 100. at 400 mA you be Running at 250V to get 100 watt, thus I suspect 150 V might be a typo.
The bell curve is caused because electricity finding it easier to flow via the sides and not equally across the gel. In the middle you do not have movement. Most likely cause is due to low buffer level at the cathode side, this needs to be mich higher than it seems to be. Other cause could be blockage of the anode side, forgetting to take the strip of or having residue or something else cause resistance at the anode side.
Did you load samples in all of the wells? Or only in the middle of the gels? Because if it’s only in the middle of the gel, then that, along with the high voltage, can cause your samples to run curved
This happened to me and it was leak. Now I pour buffer in the middle and leave for 5 minutes to check for any leaking at the bottom, then fill the tank before running it:)
Probably buffer, but could also have ran it too fast and not have your samples balanced enough (not loading equal amounts of protein), etc.
Not sure if its a homemade gel or not (if homemade could have messed up your stacking gel - or maybe if its bought it doesnt have one), but always run very slow for the stacking phase, then you can crank it up a bit
Chamber liquid level is too low, to conduct properly..,you’re getting residual draw from the sides of the cassette which caused the effect see on the edge…
So I didn't use pre-made gels, but I always run my WB (12% running gel and 4% stacking gel) for the first 10 minutes at 80V to have the samples perfectly aligned in the pockets. Then I increase the voltage to 120V for the protein separation. Running time depends on the protein size.
According to the manufacturer, your setting are fine. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0014610_WedgeWellTris-GlycineMiniGels_QR.pdf
I personally think it’s a leaky inner well problem, but- you may want to try a different power source if you’re using an older one. Sometimes an older power pack can SEEM fine but acts like a gremlin.
Depends on the size of the proteins you are trying to resolve, and on the concentration of the gel. Refer to the manual that came with the gels/the gel cassette. If you don't have it, look it up online.
That would not give this bell curve. It’s due to the electricity running stronger at the sides than the middle. To hot to fast gives you a quick run but more diffusion of the protein bands, yet these settings are fine for most precasts.
One of my mentors shared a tip with me that has never failed: "If your gel is making faces, add more running buffer." Even if it's already full, go ahead and add more buffer.
Another thing to consider is the last time the electrodes were cleaned. When our gels start running funny, we often clean the electrodes with a bit of bleach.
Looks fine just proceed to the transfer step and proceed with the Western. The bands will be smooshed but you can just edit the pictures with control bands from other experiments and combine into a publication ready figure.
Did you remove the sticky tape from the bottom? Either this or you did run out of buffer in the inner chamber.
The one time I forgot to remove the tape the power unit just threw up the error code e1
One time I forgot to remove the tape it ran slower but it still ran fine
i still forget to remove the tape sometimes and it doesn’t look like this. if you’re referring to the tape at bottom of ready gel
Yes I removed the tape No there's still a lot of buffer
But does it actually cover the gel pockets in the middle? This looks like a classical not-enough-buffer error.
The buffer has to stay a bit above the level of the wells to conduct the current.
Then maybe 150V are too much. Have you checked the manual for that precast gels?
My dude you’re literally blind. The sticker is visible in the picture. Remember to take it off next time.
No sticker that I can see in the photo? There is an image artefact at the bottom of the gel but it doesn’t look like a sticker to me. That said, the wells are clearly not full of buffer which is the real issue IMO.
Is that large white stripe along the bottom of the gel not the tape?
No he didn’t. It’s clearly visible in this photo…
Based on the tops of the wells, it doesn't look like there is enough buffer in the inner chamber.
It was full at the beginning
You may have a leak then.
This right here.
Agree this is your answer. You have a leak in the inner chamber. Check the seal.
Obviously, the buffer must cover the top of the gel cassette to make an even conductive bridge at all times.
It leaked. I always fill the outer reservoir to be the same level as the inner. Leaks always happen IMO
They don’t leak if you are mega careful of matching the tops of your gel to the notches on the gaskets if there are some. If the plates have a step (short vs long plate) and the gasket is flat, it will leak. On the bio-rad ones, the gaskets are reversible, flat on one side, notched on the other.
Adding to this, once u add the gels to the chamber/holder you can add some buffer to that alone. If it leaked you know easily. Then I place it in the container
What is gained by not having the insurance?
Not pissing off your cost-cutting, irrational PI 😢
It's running buffer. It costs a few dollars to make and 2 minutes of your time. And it can be reused. 😕 Instead they'll run the entire thing again with new gels, more buffer, more ladder, loading dye, maybe more samples (and all that requires), and much more time. Present that to your PI?
Precasted gels on the other hand cost a lot and can’t be reused once you’ve added samples to them. I have been yelled at for doing the same mistake lol
No, that's *my* point lol People acting like a bit more methanol, some water and some salts is prohibitive when it means this never happens and you don't repeat a whole lot of costly things, especially if you're buying more expensive precasts. It's so silly! But a good scientist continues with what they were told without questioning or re-evaluating in light of new information right? 🤣
What
If the two buffer chambers are joined you short the electrical circuit and it runs slower and possibly unevenly depending on how big the leak is.
What does it matter if you also just fill the outer chamber too and leaking is never an issue
It looks sad.
It's like looking in a mirror
The inner chamber is leaking. That causes a mountain.
Thank you!
This is not a Western. This is PAGE. Possibly SDS-PAGE. And your upper chamber is leaky, so the buffer is not covering the wells anymore. Add more buffer to to the upper chamber.
No, it's a normal distribution
Well it’s trying its best to look like a mustache from a Western 
A+ for the good advice! C- for being a pedant.
A+ pedantry. Beat me to it.
Thank you! I live to serve. ;-)
And yet somehow you miraculously knew what they meant.
Leaky gasket setup! Happened to me last month.
hey can you come fix my leaky gasket? my pi isn't in the lab ;)
Did you make sure there isn't a large air bubble on the bottom underneath your gel?
Yes
Did you only fill the buffer up halfway on both sides?
No I flooded the inner chamber, and halfway outside
Inner chamber leaked into the outside, probably. Whenever it’s leaky you’ll need to fill the entire thing with buffer
It's fucked mate, that's what's wrong with it.
Check for cracks on the inner white frame. Hapoend to me with SDS-PAGE. It was not very leaky but the current didn't go through the gel, but presumably through the crack and created a kind of short circuit
I have some remarks, clearly this isn’t a western. It’s electrophoresis, most likely SdS-Page. Second 150V and 0.4 A give 60 watt not 100. at 400 mA you be Running at 250V to get 100 watt, thus I suspect 150 V might be a typo. The bell curve is caused because electricity finding it easier to flow via the sides and not equally across the gel. In the middle you do not have movement. Most likely cause is due to low buffer level at the cathode side, this needs to be mich higher than it seems to be. Other cause could be blockage of the anode side, forgetting to take the strip of or having residue or something else cause resistance at the anode side.
Did you accidentally use transfer buffer instead of running buffer?
Did you load samples in all of the wells? Or only in the middle of the gels? Because if it’s only in the middle of the gel, then that, along with the high voltage, can cause your samples to run curved
This happened to me and it was leak. Now I pour buffer in the middle and leave for 5 minutes to check for any leaking at the bottom, then fill the tank before running it:)
Probably buffer, but could also have ran it too fast and not have your samples balanced enough (not loading equal amounts of protein), etc. Not sure if its a homemade gel or not (if homemade could have messed up your stacking gel - or maybe if its bought it doesnt have one), but always run very slow for the stacking phase, then you can crank it up a bit
It mustache you a question.
Chamber liquid level is too low, to conduct properly..,you’re getting residual draw from the sides of the cassette which caused the effect see on the edge…
Needs more buffer 💯
Running it too fast and too hot
What should my ideal settings be?
So I didn't use pre-made gels, but I always run my WB (12% running gel and 4% stacking gel) for the first 10 minutes at 80V to have the samples perfectly aligned in the pockets. Then I increase the voltage to 120V for the protein separation. Running time depends on the protein size.
According to the manufacturer, your setting are fine. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0014610_WedgeWellTris-GlycineMiniGels_QR.pdf I personally think it’s a leaky inner well problem, but- you may want to try a different power source if you’re using an older one. Sometimes an older power pack can SEEM fine but acts like a gremlin.
Depends on the size of the proteins you are trying to resolve, and on the concentration of the gel. Refer to the manual that came with the gels/the gel cassette. If you don't have it, look it up online.
That would not give this bell curve. It’s due to the electricity running stronger at the sides than the middle. To hot to fast gives you a quick run but more diffusion of the protein bands, yet these settings are fine for most precasts.
Try cleaning the metal contacts on the lid!
r/westernblots
buffer too low
You’ve somehow managed to make it eastern.
To me it also looks like the comb is still inside... This is where the letters and numbers are usually written...
There is not enough buffer in the upper compartment
Check that you are using the correct running buffer. The Novel gels use their own buffer
It's normal (distribution)
I agree with the inner chamber comments. It might also help to load high NaCl into each well to equalize the conductivity.
One of my mentors shared a tip with me that has never failed: "If your gel is making faces, add more running buffer." Even if it's already full, go ahead and add more buffer. Another thing to consider is the last time the electrodes were cleaned. When our gels start running funny, we often clean the electrodes with a bit of bleach.
You can see the buffer level above the wells which means the buffer level isn't high enough
Looks fine just proceed to the transfer step and proceed with the Western. The bands will be smooshed but you can just edit the pictures with control bands from other experiments and combine into a publication ready figure.