developing film manually - (Sep/06/2011 )

10' exposure is quite short, you can leave it as long as you like. If you are getting no signal after 10', leave it for 1 hour or more (even overnight) and see what you get. If you get a signal after a longer exposure, your primary is too dilute or you are washing too much - 3-4 5' washes at each step should be enough.

Don't coomassie stain your membrane - either use a ladder that you can see when you transfer, or ponceau S stain the membrane (it washes off), as coomassie can block the antibody from binding.

Ok, the acetate sheet is the saran film I was talking about,

and yes, don't dry the membrane, but remove the excess of ECL as said almost a doctor, "by just lifting the membrane with a pair of tweezers and allowing the ECL to flow down into a piece of tissue paper."
He speaks english better than me.

hi guys, thanks for all your input. I have been thinking about this day and night, and after talking to my supervisor about the possibility that it is actually the samples that might have a problem after freezing and thawing I need to ask you something, wise people: do you heat your samples at 95•C every time when you load the sds page gel?
if you do and your wbs work and mine don't, that could be a reason... thanks in advance!

I heat my samples ... I have to check now if it is 2 minutes only, usually I do 5 minutes. I have to go back to my lab books for the phosphorylations.

(stupid question) your secondary antibody is "antisheep", is your primary antibody produced in sheep?

we also add blocking agent to the antibody solutions to preabsorb any antibody that may bind to the block (we use polyclonal antibodies most of the time) and limit tween to 0.2%.

as for whether your samples are the problem, run a gel and stain it. if the pattern is normal then the problem is not your sample treatment.

hello mdfenko, yes, the primary antibody was raised against sheep, as i was reading the label this morning i almost panicked thinking it said antirabbit but it was antisheep raised in rabbit phew!

it is a polyclonal antibody that i'm using too and it binded everywhere. i was preparing the tbst 1/1000 ml tween/tbs.

as a last step, i generally stain the membrane after exposure with coomasie blue, not immediately after transfer. the pattern seemed normal to me, but then i was trying to think biochemically (which is particularly hard for me as an agronomist) and wondered whether heating the sample would make the binding site of the protein accesible to the antibody and that would be the reason why nothing was showing up on the film.

so today i heated the samples before running the gel, transfering to the membrane overnight and then tomorrow we'll see whether this has worked (fingers crossed)

thank you all

Hi again TJ, yes you were right I am a doc now

A few things for you.

1. you say in your last post that the primary antibody is anti-sheep raised in rabbit.... if this is the case your secondary antibody needs to be anti-RABBIT. ie. your primary antibody is a Rabbit IgG that recognises a Sheep protein. So if you are using and anti-Sheep secondary, is not going to work.

2. in a previous post you mentioned you are worried that the freeze-thawing might be a problem. As mdfenko said, if your proteins run fine is probably okay, my concern will be more in how you prepared your samples. Did you add protease inhibitors? do you have phosphatase inhibitors (in case you are looking for phosphorylation)

3. I know you said that your reagents are new, but here's a very quick-easy way to check your ECL (just in case). Add 1ml of ECL mix to an eppendorf tube, and then in the dark add 1ul of your secondary antibody (HRP conjugated). If both reagents are fine you should see actual light in the tube. This is just worth doing for the fun of it

4. finally, answering a previous post. I always boil my samples 5min at 95C. This might not make a difference, but will depend on the antibodies you are using. As you said on your last post, denaturation might expose epitopes in the proteins that the antibody might not reconise otherwise. Which brings me to my last comment. Is your antibody comercial or home-made. If the first, does it say it works for WB? Some antibodies don't work on WB because they don't recognise the denatured protein.

Don't despair, WB can be a pain in the neck sometimes, but oh the joy when they work

as almost a doctor says, if the anti-sheep is your primary then your secondary should be anti-rabbit. i surmise that your secondary is the rabbit anti-sheep antibody conjugated with hrp. if that is the case then your primary should be anti-protein-of-interest raised in sheep.

also, as almost a doctor said, check the information sheet that came with the primary antibody and see if it is suitable for western blotting (if not then you can try a western with non-denaturing page or a dot blot or an elisa).

i'm sorry about the confusion, the primary antibody is not a commercial one, we got it from another lab. in the label it reads:

"These antibodies were affinity-purified.
Please reconstitute with 50 ul of sterile water.
These antibodies were raised against sheep."

that's why i'm using antisheep as the secondary, which i got from sigma.

when i extracted the proteins i did use protease inhibitors from roche. thanks for the trick with the ecl, i'll give it a try

thanks guys

"These antibodies were raised against sheep."
It means that it recognizes a sheep protein, isn't it? I would say it is an anti-sheep, maybe
you should use an antibody raised in sheep against your phosphorylated protein , with your anti-sheep secondary antibody.