strange western - transfer turned PVDF, filter, sponge, and buffer yellow? (May/18/2006 )
Hi,
I wondering what went wrong.
I've been doing western for a while now.... usually turns out fine. But, i've been doing it like a cookbook, never really looking into why i do what i do...but now something went really wrong.
I'm repeating an experiment that i have done successfully before. Last night, it worked wonderfully.
So, i did my gel, it looked beautiful (really really proud of how the ladder was perfect!! 
)
Set up transfer as always (1X transfer buffer, 10% methanol), on ice. lovely.
30 min at 30v, 45 min at 75v, and 45 min at 100v.
When i opened the tank up, there was a white film on top.... never been there before. 
the buffer had turned yellow. open up the cassette, and the sponges are yellow, the filter paper is yellow (especially in the area of the gel), the PVDF is yellow, and, this is the kicker.... there was no ladder transfered on to the PVDF, or past the PVDF to the filter paper, and there not even a hint of protein anywhere in site. Also, the whole apparatus felt a lot hotter than usual.
So, i was wondering, if the sponge had left over salt dried into it (and i have 2 suspects who would do this).... could this have screwed me over?
why would the whole thing turn yellow? where has my protein gone?
Vetticus
Are you sure you are using 1x transfer buffer?
Just curious.
Vetticus
Could the yellow come from the loading dye in the gel?
Normally blue, but depends on pH.
The blotting buffer I use is at pH 8,3 though, so the bromphenolblue shouldn't turn yellow (only at low pH if I recall correctly).....
???
You're sure polarity was ok, so you din't blot the protein just right into the tank...
That has happened to me and my problem was the sponges, so I soaked them in 100% methanol to clean them for about 1/2 the day and then rinsed them with DI water and allowed to dry. Also make sure that when you touch your sponges you have on gloves because you can transfer oils and such from your hand to the sponge.
Hope you work everything out.
hello,
first and foremost, check your buffer stocks. everyone screws up a buffer or two at least once in their life (or, a lot more in my case). make new buffers and repeat the blot. it will probably work.
a few other things to check for...
- depending on the type of blotter you have, you might be getting rust buildup on the leads, which tends to dissolve into your transfer buffer during the run, and gives everything a lovely yellow/brown hue. it also ruins your blot. if you are getting rust, clean it off, or buy new leads from your supplier.
- are you sure you connected your wires properly? if you put the negative wire into the positive port on your power supply (and vice versa), you not only sent your proteins in the wrong direction, but you probably pitted and partially rusted one of your electrode plates. if you're using a blotter w/ platinum wires instead of plates, ignore this idea....
- lastly, did you run your chloride front off of your gel? if not, did you equilibrate long enough in transfer buffer and w/ large enough volume of buffer to remove your chloride ions. if not, chloride ions can make a mess of a transfer apparatus.
i hope this helps,
jon
I wondering what went wrong.
I've been doing western for a while now.... usually turns out fine. But, i've been doing it like a cookbook, never really looking into why i do what i do...but now something went really wrong.
I'm repeating an experiment that i have done successfully before. Last night, it worked wonderfully.
So, i did my gel, it looked beautiful (really really proud of how the ladder was perfect!!
)Set up transfer as always (1X transfer buffer, 10% methanol), on ice. lovely.
30 min at 30v, 45 min at 75v, and 45 min at 100v.
When i opened the tank up, there was a white film on top.... never been there before.
the buffer had turned yellow. open up the cassette, and the sponges are yellow, the filter paper is yellow (especially in the area of the gel), the PVDF is yellow, and, this is the kicker.... there was no ladder transfered on to the PVDF, or past the PVDF to the filter paper, and there not even a hint of protein anywhere in site. Also, the whole apparatus felt a lot hotter than usual.
So, i was wondering, if the sponge had left over salt dried into it (and i have 2 suspects who would do this).... could this have screwed me over?
why would the whole thing turn yellow? where has my protein gone?
Vetticus
Last week I did my transfer, after this I realized that the transfer buffer was brown and then turned red!!!
It's very strange, I think it's depend on the deionized water pH I used to prepare the buffer. May be the deionizing system is not working well (es. you need to change filters).
are you using a tris-glycine based buffer?
a friend once had accidentally set volts and amps round the wrong way - resulting in the glycine kind of coming out of solution, turning it yellowish brown and making a really gross burnt sugar kind of smell.......
is your tank working ok? you know how sometimes they just change their minds and correct your voltage selections!!!!! (or is that just my evil co-workers?)