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Advice to improve westerns - looking for ideas to clean up my WB results (May/07/2007 )

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Sorry for not "being here" blush.gif
I had some exams and been kind of busy studying... wacko.gif
Hi, I can give you the "protocol" i'm using for the -developing of the film- process... But first just a few things I found out and might be helpful for developing and westerns...
1. If you have an idea of the intensity of the signal you are expecting during the exposure is always helpful. What I mean is that if the signal (emittion of light) is strong then you don't have to expose the membrane on the film for too long (for example when I did a dot blot to chech the crossreaction between the 1st and 2ndary antibody the signal was very strong so by just exposing for 5 min i got very black and defined dots)
If the signal is not strong (few protein..) then you need to expose longer (exposing for more than 30min I think doesn't change anything more because that is how long ECL lasts) (I expose for 30 min because I have very little protein and the signal is weak)
2. I think the fixer solution (mine has to be diluted as well and I keep it in plastic bottle diluted) lasts longer than the developer but when you are doing a new kind of western or developing (that you are not sure what you will get) I suggest that you change your developer solution after 3-4 times you used it (oh, and keep it prefferably in a closed bottle).
3. Be very sure that your film wasn't exposed in light. If it was then it will come out black no matter what. When I started developing a photographer offered me a good tip to check the "dark room" i was going to use: he said to place pieces of film in different places in the room and under the "red lamp" and a coin on top. After develop and see if there is any light coming in the room. if there is light you will see the shape of the coin as transparent (no light) and the rest of the film back (light).
4. The developer solution is to "bring out" the changes that light causedto the film and the fixer is to maintain the "picture" on the film (even when you turn on the light).
OK I wrote too much...
Here is the full protocol:
1. Add ECL (if that is what you are using) to the membrane. I let it 1,5 min
2. Turn off light and (i'm using a film-cassette so I will describe it this way) put the membrane in a plastic transparent sheet (so you don't wet the film) and that in the cassette and on top the film. Close the cassette and expose for Xmin.
3. Take out film and put it in the developer solution (which could be in a plastic container) for 3 min and during that time shake gently.
4. Take the film and just dip it and wash it out in a container with water
5. Transfer the film in fixer solution for 3 min shaking it gently.
6. Again in another container with water and now you can turn on the lights
7. Hang the film to dry
8. Return the solutions to the their bottles.

This is what I do. I hope it will work for you. Sorry for all the bla, bla but when I started out westerns I had a lot of problems and I wish I had someone to tell me all that I found out the hard way. Anyway this forum was really helpful for me and I hope I was helpful too.

Good luck wink.gif

Hi Erotic terrorist!
If I understood correctly the 3 min thing worked out for you...Great! biggrin.gif
It is true that I wash a lot.. but I got that tip from a friend who does tones of westerns and on top I get no background so...i stuck with it.
Hope your blots are coming out great now. biggrin.gif Let me now...


Hey Charis, Yep I've been doing somemore recent westerns and things are working much better. No one knew about the 3 minute thing. like i said my washes are 2 quick washes, 1X 15 minute wash followed by 2x 5 minute washes. But for the hell of it I made it 4x 5 minute washes and suddenly it became much much much cleaner. so woo hoo!!

Just a note on exposing the membrane, I've got good results for even a 5 second exposure. I personally prefer to start with a 5s exposure and then estimate from there. If it looks weak then I expose longer. e.g. 3 minutes, 5 minutes or even 10.


Dear Charis: Thank you so much for your bla bla.. I really appreciate that.. I'm going to continue my western once I receive a new ECL reagent (the one I used was actually expired) and for sure, your tips will be taken into consideration smile.gif

Dear Erotic Terrorist: Thanks for your advice as well. That worths a try once I get my new ECL reagent


Erotic Terrorist:
I'm very happy that everything is working better biggrin.gif It is nice that I was some help...
Don't be afraid of the washes, they are the ones that ensure that you don't have any "leftovers" in your blot that may cause background noise or bad picture (like lets say not good enough washed out 2ndary Ab)
Hope everything continues to work out fine wink.gif

Hope you get good results and when you receive the ECL and perform your blots let us know. wink.gif
Good luck tongue.gif


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