19 replies to this topic

[toejam]

hi guys,

i'm back from durham, where we used to have a developer machine that processed the film and then everything was fine. in my lab we don't have one of those, so i have to do it manually. there's no one around that can tell me how to do it so i gave it a go on my own, a postdoc from another lab gave me indications as follows:

1. put the film in the developer (1:9 dev - water ratio) for about 1 minute
2. put in water
3. put in fixative at least 5 minutes
4. put in water
5. air dry

i tried that but there's nothing on the film. did it twice, first exposure 1 min, following 5 min. now i'm trying with 10 min.

i know it's kinda difficult to ask someone to "show" me on the forum, but if any of you has any ideas it would be great. thanks in advance! cheers.

[bob1]

It will depend on which developer solutions and film you are using as to the exact times etc.

Did the film develop at all?  If so it should have gone from (probably) a greenish or yellowish translucent sheet to a grey or blue but transparent developed film.

Generally the developer solution shouldn't need to be diluted unless the stocks have not been made up (in which case, they are usually made of several components and come with instructions)

If you know roughly how long it took for the film to come out of the machine, then you can estimate the times to use in dev,fix and water by dividing by 3.  You can't over-wash or over-fix, so don't worry about that too much.  You can agitate the film to speed up the process somewhat.

For kodak RP-X-OMAT developer/fixer solutions I have found that 30 sec-1 min in developer is enough, followed by a similar time in fixer and water works well.

[BioMiha]

Do you have a red light in the darkroom? If so, you should develop until you see spot/bands on your film. How long did you expose the film => you can increase the exposition time or use a different ECL substrate.

[toejam]

hi bob and biomiha, thanks for your input. probably one of the problems was that i diluted the developer 9:1. the film did turn transparent blue when i put it in the fixative but not before that. i do have a red light in the dark room. i exposed the film for various times: 1, 5 and 10 min but i couldn't detect anything in either of them, not even the recombinant protein. the ECL substrate is new so that should be fine. i'll try again by not diluting the developer.

thanks for your information. all the best.

[laurequillo]

Ey TJ, I used to do it manually, and normally it didn´t take long in the developer to see the bands. I used to shake it a little bit till I saw the bands, (there you can play with the exposure time) and then just move it to the fixer til it was clear and then in water. I think we had to dilute the developer and the fixer but not in those proportions

[toejam]

hi lq,

i think i got the developer thing working well now. but i'm still having problems. i put the recombinant protein and that didn't show any signal either

i've done the following:
1. run sds page gel
2. soak membrane in methanol 10' then to transfer buffer 20' shaking
3. prepare sandwich
4. transfer
(up to here i know everything goes well because the membrane stains when i finish with coomasie blue)
5. block in 5% milk + tbst (i've tried both, 1 h and overnight)
6. put primary antibody (i'm using a concentration of 1:3000) in either 0.5% or no tbst for 3 hours
7. 3x washes with 0.5% tbst 5' each
8. put secondary antibody (antisheep). have tried 1:30000 and 1:15000 (none worked) for 1 h
9. 2x washes with 0.5% tbst and 3x washes no tbst 10-15' each
10. put acetate in cassette, then membrane on top and fresh ECL 1' to incubate
11. i've tried different exposure times, including 10' exposure in cassette
12. develop - but then nothing shows on the film

what pisses me off is that, as you well know, it's a lot of work and takes about 2 days, and not to see anything (not even the recombinant protein or the shape of the membrane on the film) is quite upsetting.

anyway, i'll be using different membrane and film this time. hopefully that'll fix the problem. thanks guys.

[little mouse]

is it a development problem ? expose  a piece of film to light and develop.
is it a secondary antibody problem? dot blot the primary antibody, block, wash, try several concentrations of secondary antibody.

I don't get your point 10. put acetate?
I was used to HRP-conjugated secondary antibodies and amersham ECL substrate.
I washed 3 times in TBST and twice in TBS to get rid of the tween before to put ECL substrate (1 or 2 mL directly on the membrane, without shaking, for 1 minute. Then I dried gently the membrane by putting the membrane vertically on a kimwipe paper, put the membrane between two saran film, and expose to film in a cassette, for 0.5 to 60 minutes)

[toejam]

hi little mouse. thanks for your input. what i meant in point 10 is to put an acetate sheet in the cassette before putting the membrane, then i cover it with another sheet after putting the ECL. then in the dark i put the film on top of that and expose it for various times.

probably the membrane was too wet in ECL and that was affecting.

i will try your suggestions. thank you very much.

tj.

[almost a doctor]

Hi tj.  Is this a new protocol you are trying to do or a western that used to work and has now somehow stopped?

If the first, I'd suggest you optimise both primary and secondary antibodies by doing a dot blot to your recombinant protein.  That way you can try different concentrations without having to spend 2 days.  You could dot your protein and the cut the membrane in different strips to test different concentrations.

Regarding the ECL and development.  I also suggest to rinse your membrane in TBS (no detergent) before incubation with ECL.  Also, I understand from your post that you place the membrane WITH ECL on the cassette for development. I would incubate the membrane with ECL for 1-5min (depending on the manufacturer recommendation) and then remove the ECL before placing it in the cassette.  You can do this by just lifting the membrane with a pair of tweezers and allowing the ECL to flow down into a piece of tissue paper.

If it is a protocol that has somehow stopped working, I'd check all the reagents, specially the pH of your buffers.

Hope this helps.

[toejam]

hi almost a doc (if i remember right, you're already a doc, right? )

i learned how to do western blots in a different lab about 1 month ago, then came back to mine and tried to repeat everything. in the first lab it was kinda working and had already optimised the concentrations for primary and secondary antibodies (or so i thought), but probably doing the dot blot would provide better results, since i didn't do anything of that in the first lab.

i do put the membrane with ecl on the cassette for development. i had been told not to let dry the membrane at any point all the reagents are new, including the stock solutions that i prepared myself, however i'll check the ph of the buffers again.

thanks for your advise all the best.

tj.

[bob1]

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.

[little mouse]

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.

[toejam]

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!

[little mouse]

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.

[mdfenko]

(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.