If so do you know the answer to this Q:.
I was told by the rep that if I dry my membrane after staining with my secondaries, I will get a better signal, however I would not be able to strip and reprobe? does that mean I will also not be able to just reprobe?? I use nitrocellulose membranes
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10 months ago
11 comments:
Yeah once your membrane has been dried, it will be funky forever after if you try to reprobe. We always just scan our Westerns on the Licor when they are still wet with TBS-T. Licor is pretty sensitive but doesn't amplify like HRP does, so if you're getting bad signal you might need to load more protein rather than try to get a few fold improvement by drying the blot...
We load 50-200 ug/lane, depending on what we are looking for.
in my experience once the membranes are dry they are done. You can't do anything else with them but read the signal....
I usually did HRP as secondary and then improved either loading size of protein, first block or time exposed to HRP... sometimes even the "developing time" although I wouldn't recommend that as much since the filters tend to be very dark.
Good luck!
Thats what I figured, I had enough protein as I could see bands when I increased the scanning intensity up to 3.5 from 3.0. I'm not sure if fiddling with intensity actually increases the sensitivity of the machine or not. I've downloaded a bunch of the material off their website to try and understand it better. I HATE using equipment that I don't know the ins and outs of...
Arlenna - when you say the licor does not amplify the way HRP does, do you mean that chemoluminescence ampliflies the signal more than a fluorescent secondary? OR are you referring to the ability of the licor ABs to amplify?
I haven't used Licor and don't use nitrocellulose but I've found that I get awesome results with PVDF/HRP if we re-wet the membrane with MeOH for 5-10s after the proteins are transferred. We don't do a separate blocking step and the primary and secondary abs are diluted in PBS/casein. Stripping and re-probing work perfectly and I've used the same membrane 3-4 times (depends on the abs of course).
PiT - I prefer using PVDF membranes as you can dry them prior to applying your primaries / secondaries which reduces the background etc. Apparently, PVDF membranes create a shitload of background on the Licor system.
Yeah - what I meant to say was that we re-wet with MeOH, dry the membranes and then probe them dry. I haven't used nitrocellulose since the late 90's when the membranes were very fragile and PVDF was a much better option.
I'm not at all familiar with the Licor system. I know that we had trouble with the dry PVDF membranes using the Typhoon scanner as the membranes supposedly autofluoresce when dry. I'm sticking to HRP, chemiluminescence and dry PVDF!
Crap I was going to try dry PVDF next week. URGH...I like the Licor as I can probe for the AB I used for my Pulldown and for my protein of interest at the same time...it has worked before so I really don't know what happened now. I think the key is the amount of protein which is so hard to figure out as I'm using embryo preps.
I didn't try the Typhoon scanner, but one of the other postdocs in my postdoc lab did and all he got were black images from the autofluorescence on the dried PVDF.
Yeah, I mean how the chemiluminescence can amplify the signal as more product is formed. Since the Licor secondaries are just the IR dyes, as you figured out they won't do that.
I think changing the intensity is like changing the gain on a microscope camera--it should indeed increase your signal but might also increase noise/background. However, I haven't actually looked at our Licor to figure out if increased intensity refers to what your sample should be or what you want to see from it... i.e. do you increase the intensity to get MORE gain or LESS gain (as you would want if your sample was already intense)?
Hey, we just figured out how to use the Intensity feature on our LiCor yesterday. It's just like the "Gain" on a microscope camera, where putting the number higher means it will be more sensitive, and lower will be less.
We typically use an intensity of about 5 for our not-so-hot antibodies, but I think we need to turn it up. You might need to also! Maybe all the way up to 10!
Arlenna - Licor has 2 different ways to increase the sensitivity. The first Gain is in the actual scanner window which increases the sensitivity of the actually detector and I think it can only be changed from 1-5. The second increase is after the scan has been completely in the alter image window. The manual sensitivity bar will change how the computer maps the representation of each pixel to the screen, that can be changed from 1-10 or 1-8.
Interestingly on my blot, I was able to see bands in my 700nm channel when I increased the scanner gain, but not in my 800nm channel which makes me think that my 2nd ab may be off. I'm going to run a bunch of control experiments (plain lystate runs without the IP) to figure out just how much protein/ab I need to see a good signal.
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