DTT Protein Extraction

[admin]

One question which I got from an email from a fellow researcher:


Hi,
which reagent could replace DTT in Protein extraction?????? In sample buffer
?

thanks

[danfive]

BME-beta mercaptoethanol

[Aaina]

hi,

danfive is right. 2-marcaptoethanol is the replacement of DTT (Dithiothreitol / a Cleland's reagent . But i would suggest u to use DTT bcoz 2-marcaptoethanol is dangerous while DTT is not. Moreover, If you are working on a mixture of unknown proteins then some researchers prefer to use a combinition of DTT+marcaptoethanol.

[danfive]

Originally Posted by admin

Hi,
which reagent could replace DTT in Protein extraction?????? In sample buffer
?

Just learned that these reducing agents can be substituted for DTT (performance would be sample-dependent, as usual ) TBP-tributylphosphine, TCEP-tris (2-carboxyethyl)phosphine.

[admin]

thank you very helpful for all.

[walid]

Hi!
which difference between ampholytes and carrier ampholytes and his role this is my first question and the second is can I use the IPG-buffer with pH 3-10 for exemple and the strips with another pH?

[danfive]

Originally Posted by walid

Hi!
which difference between ampholytes and carrier ampholytes and his role this is my first question and the second is can I use the IPG-buffer with pH 3-10 for exemple and the strips with another pH?

Carrier ampholytes are just zwitterionic compounds that will separate into a 3-10 pH/pI gradient under isolectric separation. Yes you can use pH 3-10 ampholytes in the buffer with an IPG strip that can be 4-7, 7-10, anything that falls within the 3-10 pH range.

[walid]

I have more questions:
1-which reagent could replace chaps in protein extraction?
2- the quantity of the sample (I want it in µl and in µg) that I used it with rehydratation of strip 7 cm or strip 24 cm overnight and I must diluted it or no and which dilution is better?
3-the composition of solubilsation buffer that I can use it with all type of fruit?
4- Which best staining solution with comassie blue or silver staining with total protein extraction i.e. I don't know the type of protein if it is acidic or basic protein?
5-when I worked in extraction of total protein which type of strip is better can used it i.e. with pH 3-10 NL or another type of strip?
6-concerneing the gel which better the dabble gels i.e. stacking gel + gel de concentration or one gel?
7-the colour of the gel is not clear the problem is in staining solution or what?