No straight band-WESTERN BLOTTING

[mountain]

Some of the bands in my filter do not have a straight line compared to other bands even if they all are the same proteins. What can be wrong? One end of the band seemed to move faster than the the other end.

Thanks for any ideas!

 

[DocToxyn]

Welcome to the wonderful world of Westerns, Northerns, Southerns, etc! I can see from this and your other posts that you have many questions about this and other related techniques. Do you have a teacher/mentor/advisor there that you can go to with these? I'm not saying you can't post these questions here, but rather you might have quicker or more "hands on" help from someone you are working with/for. Sometimes it's just easier to diagnose a problem when you see it being done in front of you.

Having said that, my first statement, which speaks to many of the questions you pose (as well as many other questions you may have about science in general), is that not all the westerns you do are going to look like publication quality material. Bands will be wavy, interrupted, missing, not exactly where you expect them to be...this is part of the process and the reason one does multiple replications of the same experiment. Perhaps the lanes weren't rinsed out well enough, maybe you jabbed the pipet tip into the gel as you loaded the sample, these and other reasons could cause the problem you bring up. My suggestion is talk to someone there, do it again, and again and maybe again. Hopefully you'll eventually be confident in your results. Good luck!

 

[mountain]

Yeah, i have an advisor, but he is the worst advisor i ever seen. He does not know it that is why i have to bring it here and hopefully and luckily some of you have experienced it. What do you mean with rinsing the wells? THe gels i use are precast do they still need rinsing because we have not done it!

 

[DocToxyn]

Too bad about your advisor. Is there anyone else you could go to, another professor, an experienced technician/student, they may be able to help you out. Again if we're your only source, don't hesitate, but we can't always substitute for a knowledgeable person standing next to you.

As far as rinsing wells, maybe it's not so much an issue with precast gels (I've never used them). Basically once the stacking gel has polymerized you apply the running buffer and use a syringe/needle combination to suck up some of the running buffer and flush out the wells. It removes any remaining gel buffer and loose bits of acrylamide from the wells that may interfere with sample loading/running. Maybe you could try it anyway, it's not hard or time consuming to do. I'll take a look at your other posts and see if I can help further.

 

[Moonbear]

Too bad about your advisor. Is there anyone else you could go to, another professor, an experienced technician/student, they may be able to help you out. Again if we're your only source, don't hesitate, but we can't always substitute for a knowledgeable person standing next to you.

Agreed. I would hope that if your advisor doesn't know how to do what you need to do for your project, that he'd at least be able to suggest people who can help. It's just so much easier to diagnose problems when you can 1) watch someone else do it correctly, and 2) someone else can watch what you're doing to find out what you might be able to improve.

As far as rinsing wells, maybe it's not so much an issue with precast gels (I've never used them). Basically once the stacking gel has polymerized you apply the running buffer and use a syringe/needle combination to suck up some of the running buffer and flush out the wells. It removes any remaining gel buffer and loose bits of acrylamide from the wells that may interfere with sample loading/running. Maybe you could try it anyway, it's not hard or time consuming to do. I'll take a look at your other posts and see if I can help further.

I've never used pre-cast gels either, but any little air bubbles left in your wells will give you some odd looking results when they pop, so I'd think rinsing the wells would still be a good idea. Other things I can think of are over-filling wells, distorting the shape of the well with your pipet tip (sticking it in too far or hitting the sides...it takes some practice...make sure you steady your hand as you're doing that step), too much protein loaded, the current is too high, or the gel isn't straight between the plates, there are different amounts of other proteins in your sample slowing things down, etc.

Sometimes one side just always runs faster than the other, and there's no good explanation. If this happens regularly, you just have to insert more lanes with size markers. At the minimum, I always run size markers on both ends and one in the middle with any gel. It also helps when trouble shooting a technique to run more than one lane from the same sample in a single gel. Although you're measuring the same protein, there could be other binding proteins or splice variants or post-translational modifications affecting its apparent size on a Western, sometimes subtely, sometimes blatantly, and you need to rule this out by ensuring you get the same outcome with multiple runs of the identical sample. Controls are extremely important...always...but sometimes you just need to run nothing but controls when trouble shooting and save your precious experimental samples until you're sure you know what you're doing.

 

[mountain]

Thanks for your great help.

Right now you are my major source for help. I am facing my worst nightmare in science ever. My advisor is a very bad person. When i do something wrong then he blames on me. He is there to make things worse than helping me out of my myseries. He even gives me false infos and manipulate my results. One of the days he sabotished my work by changing the side of my filters so i could not get good signals. I can not bring this up to the faculty since they won't believe in me. This man just hates me. There is nothing i can do about it except that i hide away my samples from him so he can not destroy them when i am not there. This is the first time i meet such evil scientists like him.

 

[Moonbear]

Thanks for your great help.

Right now you are my major source for help. I am facing my worst nightmare in science ever. My advisor is a very bad person. When i do something wrong then he blames on me. He is there to make things worse than helping me out of my myseries. He even gives me false infos and manipulate my results. One of the days he sabotished my work by changing the side of my filters so i could not get good signals. I can not bring this up to the faculty since they won't believe in me. This man just hates me. There is nothing i can do about it except that i hide away my samples from him so he can not destroy them when i am not there. This is the first time i meet such evil scientists like him.

Why would your advisor sabotage your work? It's his reputation on the line too if you aren't productive in his lab. If you are having such major conflicts with your advisor, you should find a new advisor. Talk to your department chair or graduate program director about finding a new advisor, and if they won't help, your university should have an office with someone who handles issues of research ethics, usually within the office of the vice president for research (if you know for certain he is manipulating data, you can even report anonymously to that office so they can investigate and put a stop to it). You should not have to work under such conditions. Not only is it a hostile environment to work in, it'll take you forever to get a degree that way, and what will you do afterward if you can't get recommendations from your advisor, and haven't learned what you should have learned in that time? You'd be better off starting with a new project in a new lab than staying in such an environment. If you're not comfortable explaining the details with the other faculty in your department, just tell them you have personal conflicts that are making it difficult to work with your advisor.

You also might be surprised to find the department chair or grad program director knows more about the situation than you expect. They can't always come out and tell students not to join a particular lab, but they often know there are bad labs and try to hint in every way possible that students should be very careful about joining that lab (at my last univerisity, we had one lab like that, and while the faculty couldn't say anything, we'd highly recommend students talk to other students about which labs to join, and we know they all know about the long history of students who bailed out of that lab 2 years into their degree when they were following one dead-end project after another). The program director was more than happy to assist them in finding a new lab to work in when they finally realized their poor choice of mentor.

 

[mountain]

Thanks moonbear!

It means a lot to me with what you said and your understanding. I am so lost in his evilness. The day i found out that he changed my filter to make me go crazy i almost burst into tears and wanted so badly to quit and find another lab. But i kept going on with my work and finally i got enough signals. This man is not my major advisor. He is pointed out by my main advisor to help me with my work. somehow he must hate me so much that he determines to destroy my work so my main advisor will get mad at me. After he sabotished my work last week. I now have determined to have a talk with the major one and tell him everything tomorrow. I do not deserve to be treated and mentally torture by this evil and psycho man! It makes me so angry so a research field has people like him. I do myself a favour and everybody else by telling the major one his real evil face. I just have nothing to lose. no one deserves to be treated this way.
I am very glad that you know what i am saying because one of the girls i work with does not believe in what i told her. She had never heard about such low level behaviour from a scientist.