vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
Cyanogen bromide (CNBr) cleavage after Met residues is a very selective and almost foolproof method which is particularly useful when used in conjunction with selective proteolysis. Introduction of a few breaks in the peptide backbone using CNBr results in a protein which is more susceptible to denaturation and thus is a better substrate for proteases. Methionine is one of the more rare amino acids, so the fragments produced by CNBr cleavage are generally large.
During the cleavage process the Met residues are converted to homoserine lactone. The carboxyl terminal homoserine lactone is slowly converted to homoserine at pH 8.0 (e.g., during trypsinization). The cleavage is very efficient unless the Met is followed by Ser, Thr, or Cys residues. In this latter case, Met is completely converted to homoserine but cleavage of the Met-Xxx bond by CNBr ranges from 0% to 90%.
|
vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
In Gel Slice method #2:
A. Separating Gel
1. Load and run gel as normal
2. Stain with 0.2% Coomassie Blue/20% MeOH/1% Acetic
or with 0.1% Amido Black/30% MeOH/1% Acetic
3. Destain with 30% MeOH/0.3% Acetic
B. Digestion
1. Cut out bands of interest with a scalpel and place in an eppendorf tube
2. Wash at least 2X with 50% MeCN/200 mM Ammonium Bicarbonate pH 8.9
3. Use enough wash solution to cover the gel pieces; incubate 20 min at 37 C
4. Allow the gel pieces to air dry slightly - 10 to 20 min at room temperature
5. Add 10 mg CNBr/ml 70% formic to cover the gel pieces
6. Digest in the dark overnight
C. Extraction
1. Remove excess CNBr formic to a new eppendorf tube
2. Extract at least 2X with 60% MeCN/0.1% TFA
3. Use enough extraction solution to cover gel pieces; incubate 20 min at 37 C
4. Combine extractions with the excess CNBr
5. Dry CNBr/extractions in a centrifugal dryer
6. Add sample buffer and run on a tricine gel
|
DanielJ
Member
Registered: 21st Nov 01
Location: gwent, south wales
User status: Offline
|
awww fort we was gonna see sum boobs frm ur collection 
|
vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
Safety:
REMEMBER - CNBr is toxic and volatile. It decomposes to HCN. Always use it in a hood. Avoid mineral acids (HCl, H2SO4, etc.), the conversion to HCN will be extremely fast in the presence of these. It is advisable to have a couple of crushable ampules of amylnitrite handy (from Pharmacy) for first aid. Just use reasonable care with CNBr and also remember that if your Speed-Vac is attached to a cold trap, all your CNBr will be in the trap. Don't leave it for someone else to clean up.
|
OFcorsa
Member
Registered: 6th Jan 03
Location: Cheltenham, Gloucestershire
User status: Offline
|
Yeas thats very well and true but there are still no juggs on my screen!
|
vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
oi thats my work your talking about 
|
GT4Brody
Member
Registered: 26th Sep 01
Location: south
User status: Offline
|
Damn scientists where is the breastage?
|
Phil Hall
Member
Registered: 28th Sep 01
Location: Belfast
User status: Offline
|
Whats the point again? 
|
vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
[img]http://www.clubrubber.net/Photos/2002_08_31/Images/2002_08_31-25.jpg/[img]
|
DanielJ
Member
Registered: 21st Nov 01
Location: gwent, south wales
User status: Offline
|
yes plz
|
Phil Hall
Member
Registered: 28th Sep 01
Location: Belfast
User status: Offline
|
Aaaaaghhh, thats much better.
|
OFcorsa
Member
Registered: 6th Jan 03
Location: Cheltenham, Gloucestershire
User status: Offline
|
there is nothing wrong with that!
|
Lucky B
Member
Registered: 7th Apr 02
Location: Lunatic asylum. Drives: Hard & Fast.
User status: Offline
|
The new tanning salon on the High Street is doing a roaring trade.....
But some people used it more than others.....
[Edited on 10-03-2003 by Lucky B]
|
Thoday
Member
Registered: 12th Jan 03
Location: Drove kitted corsa b now standard corcs c exclusiv
User status: Offline
|
ahhhh what happened in the 8th pic? did an aging process or the devil appear?lol
|
SteveW
Member
Registered: 15th Jul 02
Location: Up in the clouds
User status: Offline
|
GOD BLESS AMERICA. At least they got something write.
Steve.
|
corsa12
Member
Registered: 26th Feb 01
Location: Macclesfield (cheshire)
User status: Offline
|
Heaven!!!! Take me there!!!
|
Corsa_Ash
Member
Registered: 6th Sep 02
Location: Newport, South Wales - WCM
User status: Offline
|
LOL @ Lucky B
|
Drew
Banned
Registered: 24th Nov 01
Location: County Durham
User status: Offline
|
quote:
Originally posted by DanielJ
awww fort we was gonna see sum boobs frm ur collection
ya would only see a tit
|
vibrio
Banned
Registered: 28th Feb 01
Location: POAH
User status: Offline
|
don;t have a pic of you drew
|
corb
Member
Registered: 24th Apr 02
Location: Calgary, Alberta, Canada
User status: Offline
|
Where did you gat this pic of all my ex's together at once?
|
