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

Hi guys, I've just recently completed a western blot and got my results. I've pretty much got the results i needed but to put it simply they are just not very nice to look at. I've included a couple of pictures of the 2 gels. Now keep in mind that the x-ray film itself looks much better than what you see in the pictures here. The scanner doesn't seem to scan these films very well.

So I'm wondering about the big black splotch (circled in red), the empty white spaces (arrow) and the black spots and flakes in gel 1 and I'm also wondering broken up bands (arrow) in gel 2.

I'm asking this to see if anyone recognises these issues and knows how to correct them?

The primary conc: 1:2000 (4 degrees overnight)

the secondary conc: 1:200000. (1 hour)

I used the ECL advance solution and exposed the membranes to X-ray for 10 minutes. Ask me if you need more details.

Any advice on improving my western results would be real cool. Thanks

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I think this thread may be useful. http://www.protocol-online.org/forums/inde...?showtopic=3235

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I think the problem is most likely with transfering and blocking step. The white dots look like air bubbles when transferred. The high background is likely to be from blocking or washing not enough.

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what about the blocking step? looks like skim milk blocking, try non-protein blocking

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For the blocking I use the ECL blocking powder provided with the solutions (1g in 50ml TBS-TW20) and is blocked for an hour RT.

my wash steps are 2x quick wash, 15min wash followed by 2x 5 minuted washes.

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Hi!
I can't be sure why your films look like that but I can offer some tips that I was given when starting western blots to get a better picture of them...
I think your antibodies concentrations are OK.
Try to be very careful when handling the films (don't wear gloves) because static electricity can appear and maybe that can account for your black dots.
You have to be sure that your film was not "contaminated" with light before exposing it to the membrane. maybe this accounts for large black marks.
How long do you put your film (after exposure) to the developer and fixer solutions? (or maybe you don't use that method)
I've noticed (when I was just experimenting with the film) that if you don't allow it to bathe in the fixer solution enough the film afterwards tends to dicolour and you get white patches like yours. (I let 3 min developer, 3min fixer)
About the broken band I think too that it might be a bubble...
I also agee that you might need to wash more. After the 2ndary Ab I do 5 washes with blocking(1,2,3,4,5min), 1 with TBST (5min) and 5 with TBS (with subsequently rising time, 1,2,3,4,5min). I get totally background free blots.
Good luck

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Hi Charis, yes I do use the developer and fix method for developing the film. I don't however count how long I soak them in developer or fixer. It's kind of look and see what it looks like then pull it out of the sol. But I'll try the 3 minutes method.

That sounds like a very thorough wash. With blocking solution even? I could try that next time. Thanks for all the advice so far people. The more the merrier.

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Hi, can Charis or someone offer some advice on how to develop a film after the exposure? I've been struggling a lot in this part. If I put the film too long in the developer, it'll turn black (I can only see the bands by holding the film against light source). On the other hand, if the duration of the film in developer is short, I'll get nothing. What I mean by long duration is actually by 10 seconds. I tried not to time the developing duration but to observe the bands appearance but to no avail since before I could see anything, the film would have gone black. I'm wondering whether it was the problem with the developer solution or it was my handling method.

Besides, the film that I got is always uneven and dirty-looking, as if the developer solution didn't provide whole surface coverage. However, I have always make sure that my film is fully immersed in the solution.
??

PS: Not sure whether I need to open a new topic......?

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Maybe you need to change your developer. Do you know how old it is?

I have had X-rays that has turned completely black, it's usually because it has somehow been exposed to light. Make sure the container it's in is closed tight and make sure you are in a dark room. All it takes is a computer monitor even on screen saver to expose the film.

I've tried Charis's 3 minute developer and 3 minute fix method and it is teh roxxorz!! When in the fix it will turn black and is very tempting to pull it our early but don't do it. It will clear up.

I also like to tilt the container with the solution + film in it to stir up the water a bit rather than leaving it to just sit there.

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When I began my western a month ago, I diluted the developer solution freshly out of the bottle and have been using that diluted solution ever since. However, the developer stock has been kept in the lab for quite some time. Maybe this has caused the problem.

Perhaps in the next round of my western, I'll prepare another new developer solution and at the same time, I can get some new developer from other lab, just to test and see what is the difference.

Thanks for the advice

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