Quick/One-step/automated Western Blot - (Nov/23/2011 )
A typical 'traditional' Western blot takes at least 3 days but are there any devices/equipments which allows quick protein expression analysis (substitute for the traditional quantification, separation, transfer and staining process)?
I've seen companies promote a "one-hour" detection system etc (http://www.genscript.com/western_tech.html) but is there something like a PCR machine out there yet?
science noob on Wed Nov 23 13:05:20 2011 said:
A typical 'traditional' Western blot takes at least 3 days
That sounds a bit overboard to me. I do WB from sample prep to imaging in one day following the traditional protocols. However, I know that Millipore advertizes a quick WB set, where you use a vacuum pump to suck the solutions through the membrane and shorten the incubation time. I have thought about trying it, but have not found the time (or samples that I don't cherish enough to play around with them). I also think you can easily make a do-it-yourself thing that would work just as well as the Millipore one.
BioMiha on Thu Nov 24 08:47:40 2011 said:
science noob on Wed Nov 23 13:05:20 2011 said:
A typical 'traditional' Western blot takes at least 3 days
That sounds a bit overboard to me. I do WB from sample prep to imaging in one day following the traditional protocols. However, I know that Millipore advertizes a quick WB set, where you use a vacuum pump to suck the solutions through the membrane and shorten the incubation time. I have thought about trying it, but have not found the time (or samples that I don't cherish enough to play around with them). I also think you can easily make a do-it-yourself thing that would work just as well as the Millipore one.
One day WB = 6 AM to 6 PM?
Total protein extraction (15 min - 2 h depending on how many samples you have), total protein quantification using BCA method (30 min), SDS-PAGE (3-4 h), transfer (2-3 h), blocking (1 h), primary (1h), secondary (30 min), total washing (30 min), visualise (15 min). Sounds plausible to me. Problem is I deal with large sample sizes
SDS-PAGE 1 h, transfer 1 h
What voltage do you run your gels at? and at what percentage?
I generally run 8-12% gels which take >2 h just to get the dye front off the gel @ 100V. I'm fearful of overheating if I go beyond that.
And do semi-wet transfers take shorter time vs wet transfer? I always do wet transfers.
I start at 100 V for as long as it take for the front to migrate through the stacking layer and then crank up to 200 or 250 V until the dye front reaches the bottom. The gel percentage varies with the size of the proteins I want to separate, but in most cases I make a 9 % gel. I always do a wet transfer at 200 mA for 45 min.
wow 200-250 V. Is that done in an ice bath/cold room? Haven't actually over-cranked the running voltage to that high. Max. was 120 V and I'm already sweating.
I should probably try out your running/transfer settings. it could really save alot of time albeit being in the lab the whole day.
Do you find that an overnight transfer gives better results vs. hourly transfers?
And, overnight primary antibody incubation at 4 degrees C vs. 1 h RT incubations?
If I'm worried about overheating I place the electrophoresis chamber in an ice bath. I have never had any serious problems. I never do overnight transfers, because in my opinion that is overkill. I always do Ab incubations at RT - if you think about it, most Abs work optimally at 37 degrees, why would you do incubations at 4 deg. The protein is already denatured, what do you think will happen to it?
I do my overnight transfers at 20V instead of 100V. Thing is, I'll never know which is better since I do an overnight transfer only if I'm nearing the end of the day and have to head off somewhere. Because sometimes my Western blot experiments only start mid-day to afternoon if I were to treat my cells then extract total protein.