Western Blot, what's so annoying about it

[aegistitan]

I'm about to learn western blot so I can become a western blot monkey for a lab, but I hear people saying that Western Blot is their most hated experiment. What's so annoying about it? Any tips on how to overcome these annoyances?

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[Earl Simmons]

takes forever. gel breaking when transferring it. list goes on.

 

[Narmerguy]

It takes a long time, doesn't always work, when it does work it's not always easy to interpret because sometimes it's ugly, and you're constantly deciding between just doing it again and hoping that it works better or just running with what you've got because ultimately you have a lot of other experiments to get done.

 

[Spirit of the Student Doc]

It takes a long time, doesn't always work, when it does work it's not always easy to interpret because sometimes it's ugly, and you're constantly deciding between just doing it again and hoping that it works better or just running with what you've got because ultimately you have a lot of other experiments to get done.

What I find hilarious is how these got there name (look up Dr. Edward Southern if you're interested)

The Southern blot is used for DNA analysis and was routinely used for genetic fingerprinting and paternity testing prior to the development of microsatellite markers for this purpose. The procedure is also frequently used to determine the number of copies of a gene in the genome. The concepts of the Southern blot were used in the development and creation of the modern microarray slide, which is an extensively used experimental tool.
The northern blot is a similar procedure for RNA, playing off the Southern name.
The western blot is a further pun on the Southern blot, but is an important research tool in protein detection.
http://en.wikipedia.org/wiki/Edwin_Southern

 

[Goro]

Whole lotta hard work over a 1.5 day period for something that ends up looking like this!!!

I'm about to learn western blot so I can become a western blot monkey for a lab, but I hear people saying that Western Blot is their most hated experiment. What's so annoying about it? Any tips on how to overcome these annoyances?

 

[Spirit of the Student Doc]

Whole lotta hard work over a 1.5 day period for something that ends up looking like this!!!

My first reaction was that looks like a dental x-ray. If I recall these are automated now (in a clinical setting).

 

[Spinach Dip]

Whole lotta hard work over a 1.5 day period for something that ends up looking like this!!!

You mean they're not SUPPOSED to look like that?

 

[Ismet]

What I find hilarious is how these got there name (look up Dr. Edward Southern if you're interested)

The Southern blot is used for DNA analysis and was routinely used for genetic fingerprinting and paternity testing prior to the development of microsatellite markers for this purpose. The procedure is also frequently used to determine the number of copies of a gene in the genome. The concepts of the Southern blot were used in the development and creation of the modern microarray slide, which is an extensively used experimental tool.
The northern blot is a similar procedure for RNA, playing off the Southern name.
The western blot is a further pun on the Southern blot, but is an important research tool in protein detection.
http://en.wikipedia.org/wiki/Edwin_Southern

SNoW DRoP!
Hooray Step 1 mnemonic useful for approximately 1 question.

 

[Silverflash]

SNoW DRoP!
Hooray Step 1 mnemonic useful for approximately 1 question.

I just memorized this. I can only assume I'll ace Step 1 now.

 

[ShabbaRanks]

I had no idea western blots were so dreadful!

There have been days in my lab where I run 4 gels! The 15+ well gels are what I dislike most

Western blots can suck and seem extremely time consuming initially, but after a while they become a pretty basic technique

 

[Doug Underhill]

Western blots are pretty simple. You want an annoying technique-try backscatter interferometry without an automated injection system.

 

[DenTony11235]

I'm about to learn western blot so I can become a western blot monkey for a lab, but I hear people saying that Western Blot is their most hated experiment. What's so annoying about it? Any tips on how to overcome these annoyances?

Blot comes out bad. Was it bad primary? Bad secondary? Did I not do the Bradford correctly? Maybe lysates were bad? Gotta rerun to check for each problem...


Posted using SDN Mobile

 

[deleted407021]

I personally dislike anything having to do with a vertical gel station. Leaking...leaking everywhere.

 

[Omppu27]

takes forever. gel breaking when transferring it. list goes on.

If this becomes a constant problem, then you're really doing something wrong/don't know what you're doing.


I actually like running gels. However, I work full-time in a lab and much rather have a lot of gels to run than do culture all day long.

 

[aegistitan]

Guess I picked a bad experiment to become a monkey for huh?

 

[Lake Wobegon]

See my posts in this thread lol:

http://forums.studentdoctor.net/threads/do-i-have-to-do-research.1110803/#post-15961874

 

[Lysinee]

Guess I picked a bad experiment to become a monkey for huh?

The PI appreciates your free labor. PI is happy that he doesn't have to have a post doc waste time redoing western blots now that you are here. In return, you will get a letter of recommendation when you apply.

 

[Lake Wobegon]

I personally dislike anything having to do with a vertical gel station. Leaking...leaking everywhere.

Ahhhhh.... good memories with my lab buddy....

DUDE WHY IS OUR APPARATUS LEAKING?
I DON'T KNOW, YOU LOCKED THE GELS IN!
*Rebuild the apparatus*
Hmmm, how does the SDS level look?
Stable!
*After pipetting samples*
Dude it's leaking again.
****.

You'll be fine, OP. It's not too awful. Just pay close attention to what you're doing.

 

[deleted407021]

Ahhhhh.... good memories with my lab buddy....

DUDE WHY IS OUR APPARATUS LEAKING?
I DON'T KNOW, YOU LOCKED THE GELS IN!
*Rebuild the apparatus*
Hmmm, how does the SDS level look?
Stable!
*After pipetting samples*
Dude it's leaking again.
****.

You'll be fine, OP. It's not too awful. Just pay close attention to what you're doing.

The worst part is sitting with your thumb pressed uncomfortably hard on the top of the sandwich plate for 30 minutes while your gel polymerizes.

 

[Lake Wobegon]

The worst part is sitting with your thumb pressed uncomfortably hard on the top of the sandwich plate for 30 minutes while your gel polymerizes.

All my gels were premade.

That sucks.




lol

 

[ShabbaRanks]

If this becomes a constant problem, then you're really doing something wrong/don't know what you're doing.


I actually like running gels. However, I work full-time in a lab and much rather have a lot of gels to run than do culture all day long.

I Couldn't agree more. I was in the cell culture room for ~6 hours today. It was horrible.

 

[deleted407021]

If this becomes a constant problem, then you're really doing something wrong/don't know what you're doing.


I actually like running gels. However, I work full-time in a lab and much rather have a lot of gels to run than do culture all day long.

 

[wuhsabee]

All my gels were premade.

That sucks.




lol

Ughhhh premade gels. I pretty much spent a whole morning measuring the protein concentrations for all 75 samples, preparing them all, and loading them on the gels to run. Came back 1.5 hours later ready to transfer and lo and behold... none of the samples actually ran. Freaked out and thought something was wrong with the machine.

Turns out I forgot to peel the little white tape off of the gel holders and have to start over. 🙁 🙁 🙁

 

[snowflakes]

When you get a bad WB, you have to decide if there's really nothing to see or if it was an issue any combination of the following: the sample lysis, sample concentration, primary antibody concentration, secondary antibody concentration, length of incubation time for primary/secondary antibodies, temperature of incubation, strength of chemiluminescent substrate, exposure time on x-ray, is the primary antibody you're using contaminated or just not working, etc. There's just so many variables to adjust. Process itself is tedious, even when things go right.

Gosh, I do NOT miss the frustration of doing WBs and trying to troubleshoot.

 

[Omppu27]

Ughhhh premade gels. I pretty much spent a whole morning measuring the protein concentrations for all 75 samples, preparing them all, and loading them on the gels to run. Came back 1.5 hours later ready to transfer and lo and behold... none of the samples actually ran. Freaked out and thought something was wrong with the machine.

Turns out I forgot to peel the little white tape off of the gel holders and have to start over. 🙁 🙁 🙁

Hahaha we don't have premade gels so I haven't ever encountered this problem. However, I don't feel bad for you because at least you don't have to make the gels yourself.

By the way, OP, red with red and black with black... Double check each time.

 

[deleted407021]

Way back in Bio I lab, my lab partner was in charge of making the 1X TAE running buffer, but she measured something wrong so that when we started running the gel, within 10 minutes the solution turned yellow and started fuming and our gel disintegrated....2.5 years later, in Biochem I, she's still making mistakes like that.

 

[3108]

Well...my Western Blot experiences have been pretty positive. Thankful that my supervisor in lab had troubleshooted everything over the years and now I just follow her protocol to get clean results 80% of the time. Difficult antibodies are probably the only main culprit.

 

[Cytarabine]

I spent a year trying to extract something I synthesized during chemistry research. Unsuccessfully. You only adopted frustration, I was molded by it.

 

[aegistitan]

I spent a year trying to extract something I synthesized during chemistry research. Unsuccessfully. You only adopted frustration, I was molded by it.

You write like Bane speaks

 

[Cytarabine]

You write like Bane speaks

certainly that wasn't intentional

 

[jqueb29]

Pretty much everything about them sucks. Waiting so long, so easy to break the gel, the apparatus leaking, etc. But the worst for me was the smell of the betamercaptoethanol when making the stupid gels.

 

[wuhsabee]

Pretty much everything about them sucks. Waiting so long, so easy to break the gel, the apparatus leaking, etc. But the worst for me was the smell of the betamercaptoethanol when making the stupid gels.

That stuff smelled like rotten eggs stuffed inside expired fish...reminiscent of an old, bloody period pad.

It got kinda addicting after a couple minutes, though.