Friday, October 12, 2007

SIP/MP sharing week 16

Hey there! Yup it's my turn once again. Time really flies. It's week 16!!!

In this entry, i will be sharing with you my SIP/MP experience. As mentioned by both Jiaxin and Ming Boon, we have been assigned to be 'supervisors' to the DRP students for about a month. Ming Boon and myself have been tasked to supervise the students doing the protease project (refer to Ming Boon's post on 30 Sep). However, the students from the other project will also be supervised by us should both Johanna and Jiaxin be busy. As much as it was interesting, I must say I was thrown off guard even though I was fully aware of the fact that they had never worked in this conditions before. Frankly, I think I know how the lecturers feel now :)

I'd like to share with you guys an incident that happened recently. The group working on the extraction of periplasmic proteins using chlorofrom shock (refer to Jiaxin's post on 17 Sep) were supposed to run their sample on a 1-D gel (think MBio, Western Blot). They had prepared their samples as per protocol (or so i thought). After running their samples on the gel, it strucked them that they did not denature their samples.

For the benefit of everyone, I shall sidetrack and explain the flow for running a pre-casted 1-D gel.

Basically, you will take out a certain volume of the samples into a new microcentrifuge/eppendorf tube. In this case, it was 10ul. After which, you will add an equal volume of a mix of sample buffer (I'm not sure what's in it but it contains Bromophenol Blue and SDS [gives the proteins an overall negative charge]) and B-mercaptoethanol. Any idea what's B-mercaptoethanol is for? It is a strong reducing agent that breaks the disulfide bonds found in proteins. (This step is done under the fume hood as B-mercaptoethanol gives off a very pungent smell.)


This image was taken from http://tonga.usip.edu/gmoyna/biochem341/bme.gif

Once you have done all that, you will centrifuge the tubes for 4-6 secs to collect all the liquid down. Then you will heat the samples up at 95°C for 5 mins to denature the proteins. Following this, the tubes will be centrifuged briefly again to collect all the liquid down. The samples are now ready to be loaded onto the gel.

The gel is removed from its packaging (the gel is pre-casted) and placed in the gel tank. All the samples are loaded in the wells, except for the first and the last wells where the protein ladder/marker will be added. You will then top up the buffer in the gel tank if needed and the gel is noe ready for electrophoresis. Yup so that's the steps done when running a 1-D gel. For those who are interested to know what happens after that, leave me a comment aite?

Back to the actual story. As I'd said earlier, they did not denature their samples . This means that the proteins will still be in their native conformation and thus affect their rate of migration in the gel, which in a way is pointless as the main principle of 1-D gel is to separate the linearized proteins based on their mass.

This will be one incident that I will not forget.

That wil be all for this week's post. To all, Selamat Hari Raya and hope you will have a good weekend. Four more weeks to go!!!! Take care and see you guys during the next campus discussion!

Posted by Shahirah Bibi Tg01, 0503174E






5 comments:

royal physicians said...

hi.so what happens after running a 1-D gel?
-sharon ang
tg02

VASTYJ said...

Hey shahirah,

Why is B-mercaptoethanol used instead of just proteases to break down the proteins?

Wishing you a Selamat Hari Raya!!

Loh Sharon, tg01

first6weeks said...

Hi, I would like to know what else could significantly affect the rate of migration of proteins in the gel.

Desmond Heng
0503179D
TG02

Distinction in Disaster! said...

hey mum,

i m pck!!! so u mean the 1D gel works exactly like Western blot? wa.. ur gel is pre-casted? i still need to prepare myself one lei.. haha.. so of the proteins are not denatured, what will happen during ur detection of ur proteins?

AI TEE
0503160D
TG01

J.A.M.M.Y.S said...

Hi all, thanks for the comments! nice to hear from each of u.:)

To Sharon Ang,

After running your gel, you will stain with your preferred type of stain. For example, the DRP students stain their gel with Coomassie Blue stain. There are other stains available such as SYPRO Ruby, silver stains etc. Different stains have various degree of sensitivity. For eg, SYPRO Ruby is much more sensitive than Coomassie Blue. Hope i answered your question.

To Sharon Loh,

Hi. Well, the use of B-mercaptoethanol is to linearize the proteins to make it straight.Remember our lessons in the mbio syllabus? hehe. proteases would chop up your proteins into finer pieces which is something you wouldn't want. Hope you are enlightened.

To Desmond
Hi there. There are 3 major factors that affects protein migration. One is size which is mentioned here. The other 2 are charge and structure. However, during sample preparation, SDS and a reducing agent are added to ensure that the charge of the proteins is net negative. As mentioned in the blog, heating the samples and addition of B-mercaptoethanol will help to linearize the protein sample thereby ensuring that the proteins have similar structures when undergoing electrophoresis. Hope your queries have been addressed.:)

To Ai Tee aka PCK
HELLO!!! nice to see u er.. ms/mr pck. hehe. Not exactly the same. it is just the initial step of separating your protein sample. In Western Blot, two gels will be run whereby one of them will be stained while the other will be incubated with antibodies for identifcation etc right? In this case, the gel will be analyzed and then excised (cut out the protein of interest) and send it for identification using the mass spec. Hope your doubts have been cleared!! :)

Shahirah Bibi
Tg01 0503174E