I'm doing western blots in my lab and when I reveal them they look weird. It's like parts of the membrane show bands and other parts don't. It's like they are incubating with the antibodies in a wrong way. I'm sure it's not the transference because I've dyed the membranes with Ponceau Red and they look just fine. For the incubation I've tried with little boxes and with little sealed bags. For the reveal, I use ECL plus and the Storm scanner, and I always get sure the ECL is all over the membrane. Any ideas why this is happening?
Hi,
How much protein are you loading. Try to load 20-30ug of protein and try. Sometimes if you load more amount of protein you encounter this kind of problem. Also make sure that your lysis is done appropriately. I hope you see good Actin and Tubulin.
Hi Rocio,
I assume that your antibody incubation in little boxes, etc, is with constant rocking so that there's even antibody distribution across the membrane.
If you use antibodies against a standard protein, such as actin, do you get the same effect or do you see bands across your membrane as you'd expect?
Do your membranes dry out at any point in your process? I simply ask because when I used to do WB with fluorescent Abs (rather than HRP antibody + ECL) any regions that had ever dried out (even if I had since rehydrated them) looked very odd after scanning and may be similar to what you're observing.
Hi Praveen! I'm loading 50ug of protein because the protein I'm looking for is in a really small concentration.
Hi Emily! Yes, the incubations are with constant rocking. I've got a similar pattern with actin. I don't think my membranes are drying, I try to be carefull with that.
Hi Enrique! This is my first question so I didn't know I could add files! I'll add them now.
Thank you all for your time!
Hi,
Ok, based on your images.
I agree with Praveen answer. On top of that I would make sure that the protein quantification is properly done. Overall, you should improve the following steps, protein quantification, sample preparation and sample loading. By the way, your proteins are running well (no smiley faces)
Additionally,
1) the blobs in western can be caused by bubbles during the transfer process.
2) Are you reusing the buffers? It looks the transfer buffer is dirty (small black dots).
I think that all. Good luck!
Regards
Thanks for the images!
I generally agree with the others' answers. Your membranes do not look completely odd, just perhaps overloaded. Make sure that as you put together the gel/membrane/blot paper sandwich for the transfer, to gently roll out all air bubbles that may be between the layers with a small roller as this may be causing some of your issues. Just make sure you do not stretch your gel around as it's contacting the membrane, because that can create some "smudging" effect on your membranes, in my experience.
Other general tips for WB from my hours spent perfecting it:
1) Don't re-use your transfer buffer. Reusing your initial PAGE running buffer is fine, but the transfer buffer it's best to use fresh.
2) If getting the perfect WB done is necessary in a short amount of time, pre-cast gels are definitely worth the cost.
3) Get rid of all bubbles in your transfer sandwich, but be careful not to stretch your gel around on the membrane as you do so, as this can cause smudging of the proteins on your membrane.
4) The final membrane rinses before development/reading are critical. Use fresh rinse buffer and plenty of it.
5) If you have an appropriate instrument available, switching from HRP/ECL to fluorescent antibodies is VERY worth it. You reduce background, you can't overload with ECL, and most importantly, you can multiplex!!!! That is, you can do your actin loading control Ab with a green-wavelength secondary and your protein of interest with a red one if it comes from a different animal species, and scan both at the same time without stripping between. This gives you beautiful, clean readouts that are stable and can be re-scanned hours later if you choose to. It's far, far better (and cheaper) than using ECL.
You do need access to a fluorescent scanner, such as a GE Typhoon or a Licor Odyssey. You can skip getting a fancy fluorescent protein ladder - just mark the locations of your normal protein ladder bands on your membrane with a standard Fisher lab marker or similar, which typically have fluorescent signatures and will show up nicely in an Odyssey scan, for example. I highly recommend if you can beg, borrow, or steal access to such a scanner!
Thank you all for your answers! I'll try loading less protein, and see what happens. I don't think there are bubbles in the transfer sandwich because I don't see them when doing Ponceau Red. Some of your recomendations I can't follow because in Argentina some things are just too expensive and we just can't get them, like pre-cast gels :(
Anyways, thank you all!
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