Gel Electrophorosis

huitzilopochtli

Hi, I'm trying to work out some problems involving electrophoretic gel separations. I have a Coomassie stained PAGE a western, southern and northern blot and two 2D protein analysis things represented by dots. See attachment.

I know what the western, northern and southern blots are but not the PAGE thing, though I guess it's some sort of reference table, nor the 2D things. If anyone can explain what they are and how I might use the information to answer questions (there are a couple of examples in the attachment) it'd really help. Thanks.

AtomicHorror

huitzilopochtli wrote: »

Hi, I'm trying to work out some problems involving electrophoretic gel separations. I have a Coomassie stained PAGE a western, southern and northern blot and two 2D protein analysis things represented by dots. See attachment.

I know what the western, northern and southern blots are but not the PAGE thing, though I guess it's some sort of reference table, nor the 2D things. If anyone can explain what they are and how I might use the information to answer questions (there are a couple of examples in the attachment) it'd really help. Thanks.

So, this is like homework? And you're asking us to do it for you?

Google or Wikipedia can probably tell you what you want to know. Beyond that, read your lecture notes.

huitzilopochtli

AtomicHorror wrote: »

So, this is like homework? And you're asking us to do it for you?

Google or Wikipedia can probably tell you what you want to know. Beyond that, read your lecture notes.

No, not exactly. And no I'm not asking you to do it for me. Also, I can't use my lecture notes because - last I checked - gel electrophorosis is not on the leaving cert biology course.

I have been using Google and Wikepedia (hence I know what the western, northern and southern blots are), this is really a last resort that someone would be able to "explain what [the unusual techniques pictured] are and how I might use the information to answer questions".

SomeDose

huitzilopochtli wrote: »

No, not exactly. And no I'm not asking you to do it for me. Also, I can't use my lecture notes because - last I checked - gel electrophorosis is not on the leaving cert biology course.

I have been using Google and Wikepedia (hence I know what the western, northern and southern blots are), this is really a last resort that someone would be able to "explain what [the unusual techniques pictured] are and how I might use the information to answer questions".

It's been a few years since my biochemistry days but I'll try to get you started:

1. PAGE is the method of separation on the basis of size - it may be proteins, DNA or RNA. Small molecule fragments migrate further and faster through the gel. This is usually the starting point of any kind protein analysis, gene expression etc. Google etc will tell you exactly what the technique is and how it's performed.

2. The resulting fragments, once separated, are stained with a particular agent to let you visualise the fragments. Coomassie Blue is used in your case (I used to use nasty ethidium bromide to look at cDNA).

3. The "ladder" in Gel A lane 1 is a set of markers of known molecular size, so you can compare your fragment sizes in subsequent lanes. This is normally done by using the markers to construct a standard calibration curve of marker size vs distance migrated in the gel, which should be linear.

Bascially, PAGE is the initial part of all the other techniques (western, southern and northern blots) to separate fragments, which then employ various different techniques to detect, visualise and/or quantify the protein/DNA/RNA fragment respectively. 2D protein analysis...I'm not really familiar with this, but I think it's just using another property of the protein/DNA/RNA fragment to further separate it from other fragments.

Taking Q. 73 as an example, it's asking you about DNA fragments, so I'm fairly sure you need to examine Gel C for the answer (a Southern blot, which analyses DNA). In saying that, I honestly couldn't tell you the answer without having a good think about it. I would've thought you'd need to know which lanes correspond to which samples (e.g. markers, restricted sample, unrestricted sample etc), but maybe not.

I'm sure there's a few hardcore biochem/molecular biology people on here, so feel free to call shenanigans on any of the above!

Eerie

There are good wikipedia articles on both - :eek:
http://en.wikipedia.org/wiki/Native_PAGE#Blue_Native_PAGE http://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresis

huitzilopochtli

Eerie wrote: »

There are good wikipedia articles on both - :eek:

There's no such thing as a "good" wiki article .

Thanks SomeDose for the concise explaination.

I'm still unclear on the 2D analysis. Wiki :rolleyes: didn't make it any clearer what the different sizes meant, nor how this information can be interpreted.

banjopaul

huitzilopochtli wrote: »

Also, I can't use my lecture notes because - last I checked - gel electrophorosis is not on the leaving cert biology course.

Yes it is!! Good bit on it in the chapters on genetics!:pac:

2Scoops

huitzilopochtli wrote: »

I'm still unclear on the 2D analysis.

It separates proteins by two dimensions instead of just one. So, a 1D gel would typically separate out by protein mass by sending it 'down' the gel; 2D would do this too, but also drag the proteins 'across' the gel by another, different, property (isoelectric point).

huitzilopochtli

banjopaul wrote: »

Yes it is!! Good bit on it in the chapters on genetics!:pac:

Haha, I wasn't counting that brief reference to the technique but technically you're correct. *tips hat*

Alright, thanks for the help all. I've given it a shot in any case.

banjopaul

huitzilopochtli wrote: »

Haha, I wasn't counting that brief reference to the technique but technically you're correct. *tips hat*

Alright, thanks for the help all. I've given it a shot in any case.

Haha yeah i knew what you meant, was just being pedantic!

jen_23

Prob a bit late but anyway
The PAGE is basically running proteins down a gel which seperate based on their molecular weight. The 1st lane on the PAGE gel would be a 'marker' which you use as a reference to tell what molecular weight the protein you ran is.
Coomassie basically just stains the individual protein bands in the gel blue so you can see them.

Hope this helped.