Posted 07 May 2010 - 01:12 PM
Things were going ok until I started to do western blot. I was told by the post-doc that the WB condition to detect our protein of interest has already been optimized. so I thought, wow, that's nice.
Except it's not really that way. =s
The membrane is routinely cut horizontally at the FORTH ｌａｎｅ (see picture) ａｎｄ ＦＯＲＴＨ lane only (I'll explain that later) for detection of different proteins. However, as I do more and more WB with the so-called optimized condition, I noticed significant background/unspecific bands. Out of curiosity, I decide not to cut the membrane horizontally and went ahead with blotting. Alas, a lot of unspecific bands present. As I did WB before, I went ahead and 're'-optimzied the condition again, ranging from buffer, antibody, yadda yadda. Later I figured it could be the antibodies, and I actually borrowed some aliquot of secondary antibodies from other groups and use them for further optimization. Why not primary antibody? Well, simply because no other groups are studying that protein. lol.
I then used did a series of experiment of our own secondary ab vs the secondary ab borrowed from others. I did secondary ab control, which our own secondary ab gives a lot of unspecific bands (secondary ctrl.jpg column 1), and the borrowed secondary ab is apparently 'cleaner' (secondary ctrl.jpg column 2). Then I continued the normal blotting with primary ab with different secondary abs (primary and secondary.jpg). The targetted band is at between third and forth lane.
Correct me if I'm wrong, but I thought it's pretty obvious that our own secondary ab is giving unspecific band, and that is.. bad (I repeated that several times to verify). I brought this up to my post-doc, since from secondary control I can actually see unspecific bands appear very near to the band of my protein of interest. However, the response I get was.. 'just remember to cut ur membrane at the forth lane and you are fine. If you cut lower, then the blot is dirty. Those slight background can be photoshopped".
I talked to other members in the lab and some of the comments were ''it's fine that way (to cut the membrane). All the figures in publications also show only strip of bands, not the whole blot".
I'm aware that no one shows the whole blot in their article submission. But doesn't it has to do with scientists' integrity on the results being published? With this lab, it suddenly strucks me that a number of articles out there could be very well fabricated (be it photoshopped or whatever). Oh, apologize.. I've sidetrack. Then I finally brought this up to my PI (he is very busy and seldom come to the lab) and he is shocked, as he doesn't know this happens. He thought it's ok all along as presented during lab meeting presentation, of course, with stripped blots.
He then told everyone to change secondary ab. And to repeat most (if not all) wb experiment.
Later that day my post-doc came to me and harshly accused me for being disrespectful to him, and that I challenged the whole lab by bringing this up to my PI. And he still think that our own secondary ab works and he will stick to it.
I cried upon being accused this way (bad emotional control, I know). Also because I'm almost disappointed with how the so-called science people works - be it post-doc or RA. I heard about how people 'photoshopped' their result but never really encounter one until now.
Now, my post-doc hates me and he doesn't let me do anything. My other colleagues think I'm trying to win favor from my PI and hate me (which I swear I didn't. I thought by trying out different antibody is just an initiative to make the experiment works. What's wrong with taking initiatives? I don't understand).
Nutshell, I'm very unhappy. Everyday I go to work waiting time to pass (which could be miserable). I read articles to spend time and at times I'm laughing to myself "oh, that could be photoshopped!" (of course, that IS skeptical. There must be some genuine results out there..) I thought of quitting but I'm worried it will reflect badly in my CV as I'm barely one month in this lab. =`(
I'm totally clueless about what to do next. I don't want to spend my day doing nothing..
Posted 07 May 2010 - 03:06 PM
Cutting membranes is normally done to blot with more than one antibody at once without stripping or to limit antibody waste. In many cases that's fine once you're sure the background is irrelevant and explainable... and in your case, it seems reasonable since the other secondary cleaned up the result, but didn't seem to change it... but photoshopping background out of an image (e.g. the clone tool) is absolutely not acceptable for any reason. It's counterproductive and totally unnecessary. Explaining background bands in papers is common practice and takes a whole one sentence; virtually no one makes a fuss about obvious background bands that aren't affecting the results or conclusions. If your boss was "shocked," it was probably about the shopping.
If a better 2ndary gives cleaner bands, there is no reason not to use it. You should probably keep using it on your own work. The obvious superiority of your images will be obvious enough at your lab meetings. Alternatively, you might dilute the old 2ndary further.
It sounds like there may have been a more diplomatic way to approach the problem (jumping right in and rocking the boat can cause issues), but confrontations in labs are sometimes unavoidable.
I'm not gonna pretend to know what you should do, but maybe you could work with your PI to get a project you can work on yourself, so you don't have to rely on the post-doc. And remember, post-docs move on. He'll be out of your way before long.
Posted 08 May 2010 - 04:21 AM
As far as your problem goes, i agree that its not a good idea to leave now and i second what jake says.. Now that the PI is really shocked he will or rather shud know your importance (be positive!!! ) and if you talk it out with him, may be u have a good future ahead in the same lab... and once the post doc goes or even before that noone will have the time to hold on to this and do nothing else!!! so just give it sum time and also do sumthing abt it like talking to your PI and i hope all ends up fine!!
P.S. I am proud of people like you if that is some relief!!!
Posted 09 May 2010 - 12:45 PM
Of course we should always be skeptical, but remember that the vast, vast, majority of scientists are not out to make fraudulent claims. It just so happens that you found yourself personally involved in a not so nice situation. Regarding your situation, I would make sure you communicate your thoughts to your PI about the atmosphere working with the postdoc. Maybe he can transfer you to another postdoc in the lab.
Posted 11 May 2010 - 04:43 PM
Several journals that I know of are now asking for images of the whole blot to ensure that the band you are talking about is actually the result shown in the paper, and to ensure that no photoshopping other than cropping the image has take place (there are very good tools for detecting this).
It is however, relatively common to cut a membrane below a certain size to remove background that may drown out actual signal by being so bright that the film/detector won't pick up the specific bands easily. I have heard this is done for genes such as BRCA1 quite often.
Stick to your guns on this one, people will forgive and forget with a bit of time. It may pay to keep your head down and just do your work as much as you can for the next month or so. If you still have real issues after a few weeks, try talking it over with the post-doc by getting someone neutral (another PI, head of department, senior technician) to act as a mediator during a meeting.