can a pellet be called "probe"? - (Mar/18/2006 )

sorry for the question but im confused. in the protocol i found about protein purification it says centrifuge and store the supernatant. then denature the probe... ....since there is nothing other than the pellet i am assuming it is the pellet itself...cant figure out why it is called the probe though...

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hi
i don't think a pellet can be also called a probe. May you tell your protocol completely?
What is the purpose of this protocol?

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Could it mean that you're to prepare whatever your going to presumably probe the pellet (or supernatant) with?

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this is exactly the protocol...

Protein purification
1) under native conditions

spin 1.5 ml of induced culture (5 min at 14,000 rpm)
resuspend pellet in 200 µl PBS and centrifuge ca. 5 min at 14,000 rpm
resuspend pellet in
100 µl PBS, 0.5 µl PMSF (100 mM)
Lysozyme (final concentration 100 µg/ml)
2 µl 0.5 M EDTA pH 8.0
20 min at 4°C
3-4 min sonication bath
spin 5 min at 14,000 rpm
take supernatant and store at 4°C

denature probe
5 min at 50°C
2 min at 100°C
put 4 µl probe 4 µl H2O 4 µl 4x Laemmli Sample Buffer on the gel

thank you for your help

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hi
i don't hink that it's the protein the probe as you purify it in native conditions and you have the laemmli sample buffer that is denaturing. My idea regarding this protocol is that you're doing a far western.
Target protein is separated in gel, and the interacting protein complex is added after?...
z

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fred, im still confused sorry, i think laemmli buffer is added to examine the pellet and the supernatant on the gel... so actually as my protein is supposed to be in the supernatant it is purified under nature conditions, since only small portion will be treated with Laemmli buffer for loading....
but what you mean is that the interacting protein complex is called a probe.... ....please can you clarify further...thanx a lot.

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What is the goal of the experiment? What piece of information are you trying to determine with use of the protocol you listed?

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i am purifiying my protein out of E coli ....growing E coli, inducing and purifying.....am I confusing something? i am just purifying and load on SDS-PAGE to see how much of my protein is in soluble fraction...before attaching it to the sepharose beads (GST) .....

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A probe is something that is used to detect something else. (like a fish hook)

I am sure you are supposed to have the probe in a completely different tube, if whatever you are fishing for is in your supernatant.

You obviously will have to rethink what you are dooing. Make a drawing of the protocols including all tubes with arrows between them. This way it's easier to understand and remember the protocol.

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Okay, I see the growing, inducing, and lysis parts, but where's the purification part?

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