Western blotting trouble shooting thread

cupid_s

Not long ago someone mentioned that as so many of us PhD students seem to have problems with our westerns we should set up a help group!

Well I am now in really desperate need of some help.

Our blots haven't worked for about 6 months and this work is crucial to our PhDs.

I did one yesterday/today with samples which I have detected the protein in question in previously, and also some new samples. I did this one with complete new solutions for everything.

Stained gel with coomassie blue and got beautiful bands. But after transferring and staining again with ponceau red nothing appears!

I am transferring for the same time I always have done (1 hr) but have also tried increasing and decreasing the time to no avail. So I am sure I'm not under/over transferring but if not where are my proteins going?

point3

cupid_stunt wrote:

Not long ago someone mentioned that as so many of us PhD students seem to have problems with our westerns we should set up a help group!

They probably didn't mean set it up on a money-saving chat forum :whistle:

cupid_s

Firstly - how else can I get the opinion and help of people on this board who I know have more experience in this than me

Secondly - this is actually money saving related. If I don't do this work, I will really really overrun my PhD. Meaning I have no funding for months and months, can't pay mortgage, have to spend all my (currently almost non-existant) savings on living and get into lots of debt.....

free_radical

email me on and I can help you.

Although I have left the world of western blotting far behind I do know some god tips!

misskool

can you list out your transfer protocol with your buffers and equipment? that might help us see if there's anything obvious....

A good molecular biology forum I use is http://molecularbiology.forums.biotechniques.com/forums/index.php
although it appears to be down at the moment.

cupid_s

i'll do all that tomorrow but i'm still at uni trying to sort it all out at the moment and i just want to go to bed now

i do hope it's something stupid we're doing

cupid_s

ok
this is what i've been doing.....
I have been using a 10% resolving gel and 4% stacking gel. I have been loading 15 microlitres of sample (as well as protein standards obviously) and running the gel at 100V for about 1hr 30 mins (using a 10x running buffer pre bought).

I stain the gel with coomassie blue (in methanol/acetic acid/H2O) overnight and get beautiful bands and then destain for about an hour (in methanol/acetic acid/H2O).

Make my presoaked sponge/filter paper/membrane/gel sandwich in my cassette and transfer for 1 hour at 250mA (using a 25x transfer buffer pre bought). Then I try staining my membrane with ponceau red and there's nothing there.

Any ideas or questions?

misskool

Since you've bought the solutions, are they from the same company? You can ask them why it's not been working. Invitrogen have a great support website.

Also, you'll probably thump me over the head for this one but I made the mistake before, have you put the casette in the correct order? Could you be transferring the protein into your buffer?

cupid_s

misskool wrote:

Since you've bought the solutions, are they from the same company? You can ask them why it's not been working. Invitrogen have a great support website.

Also, you'll probably thump me over the head for this one but I made the mistake before, have you put the casette in the correct order? Could you be transferring the protein into your buffer?

the solutions are the same ones as the lab next door who are having no probs. In fact someone from that lab is currently transferring a gel of mine just to see if they can get it to work.

I am definately loading the cassette correctly. I thought this might have been my mistake but i'm not doing it wrong.

One thing though. When we stain the gel with coomassie blue, obviously the protein is stained so when we transfer it the blue bands should transfer to the membrane also. Am i correct in thinking this?

misskool

I don't stain my gel with comassie blue so I wouldn't be able to help you

but if you load a prestained protein ladder, you should see it transfer across to the membrane. We use the rainbow markers (can't think where they are from at this moment)

cupid_s

i am hoping its something as stupid as the equipment is broken because nothing, not even the ladders are transferring properly. but you can see the bubbles coming off the cassette holder and the power pack works.