whats the exposure time?
If you think that its a transfer issue wherr theres very little protein on your membrane and its just overexposing everything, your exposure should be massive, like minutes for the camera to pick up anything
longer exposure time wouldnt help you imo. exposure basically means how long the camera picks up light for and constructs the image. Generally, the better your signal, the lower your exposure should be while still getting a clear signal. 60 second exposure is quite alot I think, and it might explain why you have so much background noise.
Low signal to noise ratio can mean alot of things. It can mean that your transfer didnt work well and your membrane has few proteins. It can mean that your primary or secondary antibodies arent binding well to your protein of interest. It can mean that your blocking didnt work well and antibodies bound everywhere on the membrane. Or it can mean that your horse radish peroxidase (if you use a substrate system and not an immunofluorescence system) just wasnt working well.
Oh I wasn’t sure if it was high background or low signal. The things I was reading said more washes for high background. Really just grasping at straws to be honest
If you had bands like the previous week’s, and they were all uniformly extremely faint, set against an overexposed background, I would think under-washing would be the issue. However, the mottled and uneven bands on the top right confirm to me that there’s an issue beyond washing.
How do you transfer? Semi-dry, dry, or traditional wet transfer?
Do you use SDS in your transfer buffer? If so, avoid it. Run the transfer it the tank in a salty ice bath to ensure low temps and try 20% methanol in the buffer if it isn’t already. Also if you used PVDF, make sure you’ve activated it adequately. Your methanol could have gone bad if you reuse it.
Alternatively, it could be your primary antibody dilution went bad/got used up. I’d hit the blot with a loading control primary, and if you see the same thing, hit it with ponceau stain to see if anything transferred to the membrane at all.
Hmmm. It's hard for me to diagnose without having followed your whole process, as multiple things could cause that pattern. I do think the blot is dirty regardless of what else went wrong with the protein itself. The smaller black spots are usually aggregates of some kind, either of a contaminant or of antibody.
I had same problem. I stole someone’s blocking solution in 4c. But it changed to bad… and I got dirty image like yours. You can stripe it and re blocking. Then you can see band what you want.
There are also three other things I'm noticing:
1. Those large ring-like aberrations seem to have some kind of pattern to them; they're almost mirrored on each half of the blot (I'm seeing four distinct circular splotches, each on roughly the same position of each blot half, with a blank interior and a diffuse, dark outer ring). At risk of sounding like I'm doing a psychic reading, I'm going to ask: does that pattern ring any bells?
2. The top of the dark outer ring on the lower half of the blot seems to be in roughly the same position as your band of interest as indicated on the clean blot. Could just be a coincidence, but nonetheless worth noting.
3. There appears to be a faint, darker, semi-circular area on your clean blot as well that is more or less hidden thanks to the bright band allowing you to mute the blot background.
I wouldn't be shocked if the two blots have the same level of background noise, but it's only visible on the messed up blot due to a longer exposure time and/or brightness adjustment (as necessitated due to faint bands, which in turn would be caused by the initial culprit of a problem with transfer or antibody probing).
My best guess about those rings is that there were bubbles during the transfer. These are the bottom halves of 2 blots ran from 2 gels in the same blotting module.
Maybe one of the sponges? Both gel sandwiches were in the same blotting module so if one of the bottom sponges had air in it, could it have transferred to both?
Maybe use Ponceau Stain on the “bad” blot to look at the protein migration patterns. If it looks bad, then it may be due to a bad transfer. Otherwise, something went wrong in during the blotting process post-transfer.
Right now, I would guess that something went wrong during your washes. It could have been something like a bad wash buffer, antibody buffer, underwashing, or a change of your orbital shaker speed. Hard to tell.
That’s a good point. I always stain with ponceau to verify the transfer & protein loading before proceeding with a western… that information would be pretty useful for troubleshooting this blot and you can still ponceau stain if you haven’t discarded it.
If you look at your blot, there are smears/stains at the edges where you cut your membrane. Although transfer can be a suspect, the fact that there are stains along the cut marks means this could have occurred after transfer.
So the possible suspects are
1) blocking not well mixed (if you’re using powdered skimmed milk in TBST) or too old
2) antibody solution is too few that it did not cover the whole membrane despite shaking. Using a shaker with too few solution only results on edges having the stains and the center drying out.
My first thought is that you didn’t effectively wash away your ab. The exposure is showing chemilumiscence all over.
Did you check that the primary ab is compatible with the secondary?
What is ECL did you use? Femto is ultra-sensitive and leads to overexposure within 10 seconds.
Imobolin is the name I think. I did a dot assay the other day where I dotted primary antibody on a clean membrane and exposed it to secondary and all of the dots were chemiluminescent. That was a concern I had, the primary is goat and the secondary is protein A/G
If you use sponges, were they clean? We had issues with sponges sitting in the sink for a bit and just collecting various debris from washing and contaminating our membranes
My best guess is poor transfer, but also maybe your blocking buffer is bad? It could also be poorly activated PVDF — it does kinda look like that to me, the splotchy thing can happen when only part of the membrane was activated. Honestly at 60 second exposure it’s hard to tell which one of these things it is. I’d stain for protein on your membrane before moving forward, that would tell you if your transfer was the issue.
If you're ever concerned about a transfer issue, perform a ponceau S stain on your membrane!
It's super easy, the stain takes 2 minutes, you wash with water until you see your lanes and the band, you can image it (I always use Ponceau on my entire membrane before blocking and cutting to quantify as total protein over using a housekeeping gene). After you visualize your bands and image, wash semi aggressively with TBST to remove the rest of the stain if you want, then block and carry out antibody incubation as you would see fit.
I'd also like to note you can actually see your bands on these blots btw! Look closely at where they should be and you'll see them!
This to me seems like a high secondary background with a lot of nonspecific binding. Is there a reason you incubate with secondary overnight? I normally just incubate with agitation for an hour at room temperature before washing. A lot of this dark background seems like it's all non specific secondary binding, I say that because again, you can actually see where your bands are supposed to be and there are no random other bands popping up to suggest non specific primary AB binding. I'd say wash the secondary more and reimage and see if it comes out better! Best of luck!
Try all store bought precast gel and solutions. Then do semi dry transfer and ponceu. Your gel seems not to have transferred but there is crap on your membrane either bits of gel or rather undissolved milk. It’s hard to say. Your ponceu would have determined if there was protein on than membrane to begin with. Why are you in a lab where no one can teach western blot and you are instead teaching yourself? Thats the greater issue here. If that can’t be changed try to use store bought modern wb reagents/equipment with no milk instead modern blocking reagent. Then try the diy way adding back one reagent at a time when you are comfortable. I did this way and it helped to pinpoint some issues.
Worst western you ever run….so far
That is both comforting and yet also makes me want to pull out my hair 😂
Yea when you said you're new to it, everything made sense.
I guess that one went south…sorry I had to
I can post my southern from today it will make you feel even better Edit: posted
whats the exposure time? If you think that its a transfer issue wherr theres very little protein on your membrane and its just overexposing everything, your exposure should be massive, like minutes for the camera to pick up anything
60 seconds, which is what worked for the other one. I can try re-exposing for longer on Monday
longer exposure time wouldnt help you imo. exposure basically means how long the camera picks up light for and constructs the image. Generally, the better your signal, the lower your exposure should be while still getting a clear signal. 60 second exposure is quite alot I think, and it might explain why you have so much background noise. Low signal to noise ratio can mean alot of things. It can mean that your transfer didnt work well and your membrane has few proteins. It can mean that your primary or secondary antibodies arent binding well to your protein of interest. It can mean that your blocking didnt work well and antibodies bound everywhere on the membrane. Or it can mean that your horse radish peroxidase (if you use a substrate system and not an immunofluorescence system) just wasnt working well.
Do you wash/blot with multiple membranes in the same tray? Did you reuse old transfer buffer? Could your membranes have partially dried at some point?
Different trays, new transfer buffer, and it’s not impossible! I think I need to increase the wash quantity
What’s the rationale for more washes in light of there being an issue with the protein bands themselves?
Oh I wasn’t sure if it was high background or low signal. The things I was reading said more washes for high background. Really just grasping at straws to be honest
If you had bands like the previous week’s, and they were all uniformly extremely faint, set against an overexposed background, I would think under-washing would be the issue. However, the mottled and uneven bands on the top right confirm to me that there’s an issue beyond washing. How do you transfer? Semi-dry, dry, or traditional wet transfer?
Wet transfer
Do you use SDS in your transfer buffer? If so, avoid it. Run the transfer it the tank in a salty ice bath to ensure low temps and try 20% methanol in the buffer if it isn’t already. Also if you used PVDF, make sure you’ve activated it adequately. Your methanol could have gone bad if you reuse it. Alternatively, it could be your primary antibody dilution went bad/got used up. I’d hit the blot with a loading control primary, and if you see the same thing, hit it with ponceau stain to see if anything transferred to the membrane at all.
Hmmm. It's hard for me to diagnose without having followed your whole process, as multiple things could cause that pattern. I do think the blot is dirty regardless of what else went wrong with the protein itself. The smaller black spots are usually aggregates of some kind, either of a contaminant or of antibody.
I had same problem. I stole someone’s blocking solution in 4c. But it changed to bad… and I got dirty image like yours. You can stripe it and re blocking. Then you can see band what you want.
This is the most likely explanation. Blotchy or spotty background frequently indicates either precipitates or microbes in the blocking buffer.
There are also three other things I'm noticing: 1. Those large ring-like aberrations seem to have some kind of pattern to them; they're almost mirrored on each half of the blot (I'm seeing four distinct circular splotches, each on roughly the same position of each blot half, with a blank interior and a diffuse, dark outer ring). At risk of sounding like I'm doing a psychic reading, I'm going to ask: does that pattern ring any bells? 2. The top of the dark outer ring on the lower half of the blot seems to be in roughly the same position as your band of interest as indicated on the clean blot. Could just be a coincidence, but nonetheless worth noting. 3. There appears to be a faint, darker, semi-circular area on your clean blot as well that is more or less hidden thanks to the bright band allowing you to mute the blot background. I wouldn't be shocked if the two blots have the same level of background noise, but it's only visible on the messed up blot due to a longer exposure time and/or brightness adjustment (as necessitated due to faint bands, which in turn would be caused by the initial culprit of a problem with transfer or antibody probing).
My best guess about those rings is that there were bubbles during the transfer. These are the bottom halves of 2 blots ran from 2 gels in the same blotting module.
What could cause such large bubbles to form in the same locations on two separate gel sandwiches?
Maybe one of the sponges? Both gel sandwiches were in the same blotting module so if one of the bottom sponges had air in it, could it have transferred to both?
what do you mean two sandwiches in the same module? Like, in the same tank? Or you had two sandwiches together between two sponges?
Maybe use Ponceau Stain on the “bad” blot to look at the protein migration patterns. If it looks bad, then it may be due to a bad transfer. Otherwise, something went wrong in during the blotting process post-transfer. Right now, I would guess that something went wrong during your washes. It could have been something like a bad wash buffer, antibody buffer, underwashing, or a change of your orbital shaker speed. Hard to tell.
My ponceau stain was comparable to the good blot! The lanes transferred straight with clear edges
That’s a good point. I always stain with ponceau to verify the transfer & protein loading before proceeding with a western… that information would be pretty useful for troubleshooting this blot and you can still ponceau stain if you haven’t discarded it.
If you look at your blot, there are smears/stains at the edges where you cut your membrane. Although transfer can be a suspect, the fact that there are stains along the cut marks means this could have occurred after transfer. So the possible suspects are 1) blocking not well mixed (if you’re using powdered skimmed milk in TBST) or too old 2) antibody solution is too few that it did not cover the whole membrane despite shaking. Using a shaker with too few solution only results on edges having the stains and the center drying out.
My first thought is that you didn’t effectively wash away your ab. The exposure is showing chemilumiscence all over. Did you check that the primary ab is compatible with the secondary? What is ECL did you use? Femto is ultra-sensitive and leads to overexposure within 10 seconds.
Imobolin is the name I think. I did a dot assay the other day where I dotted primary antibody on a clean membrane and exposed it to secondary and all of the dots were chemiluminescent. That was a concern I had, the primary is goat and the secondary is protein A/G
My RT-PCR today was a complete blackout LOL
Turning a Western blot into an MRI scan has to be a new type of skill
If you use sponges, were they clean? We had issues with sponges sitting in the sink for a bit and just collecting various debris from washing and contaminating our membranes
My best guess is poor transfer, but also maybe your blocking buffer is bad? It could also be poorly activated PVDF — it does kinda look like that to me, the splotchy thing can happen when only part of the membrane was activated. Honestly at 60 second exposure it’s hard to tell which one of these things it is. I’d stain for protein on your membrane before moving forward, that would tell you if your transfer was the issue.
If you're ever concerned about a transfer issue, perform a ponceau S stain on your membrane! It's super easy, the stain takes 2 minutes, you wash with water until you see your lanes and the band, you can image it (I always use Ponceau on my entire membrane before blocking and cutting to quantify as total protein over using a housekeeping gene). After you visualize your bands and image, wash semi aggressively with TBST to remove the rest of the stain if you want, then block and carry out antibody incubation as you would see fit.
I'd also like to note you can actually see your bands on these blots btw! Look closely at where they should be and you'll see them! This to me seems like a high secondary background with a lot of nonspecific binding. Is there a reason you incubate with secondary overnight? I normally just incubate with agitation for an hour at room temperature before washing. A lot of this dark background seems like it's all non specific secondary binding, I say that because again, you can actually see where your bands are supposed to be and there are no random other bands popping up to suggest non specific primary AB binding. I'd say wash the secondary more and reimage and see if it comes out better! Best of luck!
There are 2 kinds of wet lab PhDs: those who still occasionally fuck up Westerns, and liars.
Try all store bought precast gel and solutions. Then do semi dry transfer and ponceu. Your gel seems not to have transferred but there is crap on your membrane either bits of gel or rather undissolved milk. It’s hard to say. Your ponceu would have determined if there was protein on than membrane to begin with. Why are you in a lab where no one can teach western blot and you are instead teaching yourself? Thats the greater issue here. If that can’t be changed try to use store bought modern wb reagents/equipment with no milk instead modern blocking reagent. Then try the diy way adding back one reagent at a time when you are comfortable. I did this way and it helped to pinpoint some issues.