Western blotting trouble shooting thread

misskool

Another thought, what membrane are you using?
We use milipore's finest, because we're too lazy to troubleshoot and the boss has enough money to burn.

have you done the transfer in another lab? what transfer do you use? we use the bio-rad transfer with an ice block so we do at room temp. if you don't have an ice block you need to do in a cold room.

sorry, i'm sure you know it all and it's frustrating to read the same advice again and again...

cupid_s

misskool wrote:

Another thought, what membrane are you using?
We use milipore's finest, because we're too lazy to troubleshoot and the boss has enough money to burn.

have you done the transfer in another lab? what transfer do you use? we use the bio-rad transfer with an ice block so we do at room temp. if you don't have an ice block you need to do in a cold room.

sorry, i'm sure you know it all and it's frustrating to read the same advice again and again...

we are using hybond nitrocellulose membrane from amersham biosciences. it has always worked before

the transfer is being done in another lab as we speak by a blotting expert! so we can hope it'll work. we also use bio-rads transfer system with an ice-block

misskool

cupid-fingers crossed!!

we use the immobilon membrane.
http://www.millipore.com/catalogue.nsf/docs/C3117

cupid_s

didn't work!
the blue bands transferred and then were just washed off the membrane!
and no protein was actually transferred - just the dye

RoCas

Is it just me, or does anyone else not have an inkling what the hell they're talking about

misskool

cupid, it seems like the membrane is the problem. It doesn't seem to be absorbing the proteins. which hybond are you using? hybond-p?

What conditions were changed in the other lab? Maybe you can try some of their membrane?

point3

RoCas wrote:

Is it just me, or does anyone else not have an inkling what the hell they're talking about

They are doing a lab test to find proteins.
They are putting it on Martin's web-site because it will save cupid_stunt a lot of money

talksalot81

I assume we have several biologists/biochemists in here... maybe you might help.

I have transformed a plasmid using E. Coli. Now I strongly believed (as I THINK did my currently absent supervisor) that one would say 'plasmid was transformed INTO E.Coli' but I have a ref report telling me it is 'plasmid transformed IN E.Coli'. So which is it, IN or INTO?

Thanks!

CM86_2

RoCas wrote:

Is it just me, or does anyone else not have an inkling what the hell they're talking about

Not the foggiest, but wanna join me in setting up and eastern blotting process thread?

misskool

CM86 wrote:

Not the foggiest, but wanna join me in setting up and eastern blotting process thread?

No eastern blotting

but there is northern blotting, southern blotting, southwest blotting