Author Topic: basic range - general advices?  (Read 9368 times)

Vinzenz

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basic range - general advices?
« on: July 28, 2004, 05:00:00 PM »
I would like to complement my analysis of pI4-7 with the basic range. My experience comparing 6-9 and 6-11 strips was, that 6-11 does not really increase the number of reasonable spots. (I got only smears in the very basic range.)
What about the new 7-11 strips with "new chemistry"? What means "new chemistry"? Does it really give better results in the very basic range? Does it behave very different? Do I have to expect that my protocolls for 6-9 will not work?
And could someone comment on a protocoll that is not optimized for speed but for resolution and robustness (also for samples that contain a little more interfering substances).
Thanks a lot
 Vinzenz

Reiner Westermeier

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Re: basic range - general advices? (Vinzenz)
« Reply #1 on: July 28, 2004, 05:00:00 PM »
pH 7-11 IPG strips provide a better resolution up to pH 11 because a new acrylamide derivative is included, which
a) has improved buffering capacity,
b) improves the chemical stability of the basic gradient, preventing alkaline hydrolyses of the buffering groups of the gradient.
I do not know your protocol for pH 6-9, so I cannot judge, whether it will work for pH 7-11. The best is to apply the settings according to the instructions packed with the strips.
It is strongly recommended to use DeStreak in the strips and apply the sample at the anodal end of the strip.
A protocol for basic gradients is necessarily optimized for speed, because some proteins are unstable at their isoelectric points in particular at alkaline environment. If you have very stable proteins, you can run the separation over night, the "new chemistry" makes the strips stable enough.

Vinzenz

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(No subject)
« Reply #2 on: August 16, 2004, 05:00:00 PM »
Thanks for the information.
Does the following protocoll sound good? I am not sure about the amount of Chaps in the rehydration buffer.
What about adding Isoprop and Glycerol? Do they also improve running on the new gels?
Is it preferable to put IPG Buffer pH3-10 in the extraction buffer?

Basic Strips pI 7-11, 24 cm

Rehydration
7M Urea
2M Thiourea
0.5 % Chaps (or is it better to use 2 %)
0.5 % IPG Buffer pH 7-11 NL
Destreak 12µl/ml

Extraction
7M Urea
2M Thiourea
4 % Chaps
10 mM DTT
2 % IPG Buffer pH7-11 NL

Rehydrating Strips for 6 hrs
anodic cup loading 150µg in 100 µl

500V step-n-hold 1h
1000V gradient 6h
8000V gradient 3h
8000V step-n-hold 6h

Thanks for any suggestions!


Sjouke Hoving

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Re: (Vinzenz)
« Reply #3 on: August 16, 2004, 05:00:00 PM »
Hi,
Your protocol looks fine. I don't have a lot of experience with the new 7-11 gradient, but your set-up seems okay. I would increase the CHAPS to 2% and yes, add 10% isopropanol and 5% glycerol (best quality you can get). In the extraction buffer I would recommend 2% Pharmalytes 3-10 and 1% DTT.

Can you provide us with an jpeg image of one of your gels, that would be interesting.

Sjouke

Modified by sjouke at 8:10 AM 8/18/2004


Daniel A

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Re: basic range - general advices? (Reiner Westermeier)
« Reply #4 on: August 17, 2004, 05:00:00 PM »
Dear Reiner

do also have the IPG pH 3-10 NL, 24 cm strips a new acrylamide derivative? I observed that Amersham changed the package of the strips, the strips are now individually packed in separated package slots. Are the old IPG pH 3-10 NL, 24 cm strips stable in the basic pH range? Is it ok if I rehydrate IPG pH 3-10 NL strips, 24 cm overnight at RT and separate protein over 28 hours at 20° C?

thanks for your advice!

Daniel A


Vinzenz

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amount of DTT for Destreak strips
« Reply #5 on: August 17, 2004, 05:00:00 PM »
Thanks for the suggestions! Isn't 1 % DTT (65mM) to high?
In the recommendations for Destreak they say 10 mM is tolerated. Also in the discussion Josef mentioned 2.5 % is way to high and would kill the effect of Destreak.
http://amersham.zeroforum.com/zerothread?id=10820" TARGET="_blank">http://amersham.zeroforum.com/zerothread?id=10820
So I am a little confused. But obviously you get nice gels with 1% DTT.

I have not done any gels with these strips so far. But when I have I am happy to post a gel.


josef buelles

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Re: amount of DTT for Destreak strips (Vinzenz)
« Reply #6 on: August 18, 2004, 05:00:00 PM »
Hi Vinzenz, you're right, 1% is to high. The recommendation is 10 mM with anodal cuploading. Up to 20mM DTT have been tested succesful with 100ul sample and anodal cup loading. Even if DeStreak is still in large excess (versus DTT) present, there is another reaction which shouldn't be underestimated. In the basic region DTT can react directly with DeStreak, forming mercaptoethanol ! Mercaptoethanol will move towards pH 7.5 (where it concentrates) and will reduce proteins, until it's completely consumed. As you can imagine, both, the DTT concentration and your 1st dimension protocol/time will contribute to the final result. BR Josef

Vinzenz

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7-11 gels
« Reply #7 on: August 29, 2004, 05:00:00 PM »
So I performed the first gels pH7-11 as discussed above. Results are so far not satisfactory. Here is the worse gel:

Vinzenz

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7-11 gels (Vinzenz)
« Reply #8 on: August 29, 2004, 05:00:00 PM »
Here is the better gel.
Result is: Focusing in basic range is fine. But focusing in range 7 to 8.5 is bad / no focusing.
My guesses:
1. Focusing time is not sufficient
2. Chaps is interfering because it will migrate in direction of anode and accumulate.
3. 2 % Ampholytes in extraction buffer are to high and lead to higher conductivity and slower focusing.

Thanks for any advice!

Here the conditions:
24 cm 7-11 strips, rehydrated for 10 h in 450 µl of
7M Urea
2M Thiourea
2 % Chaps
10 % Isopropanol
5% Glycerol
12µl/ml Destreak
0.5 % IPG Buffer pH 7-11 NL

100µl sample (60µg for bad gel and 150 µg for better gel) applied anodical by cup loading in:
7M Urea
2M Thiourea
4 % Chaps
10 mM DTT
2 % IPG Buffer pH3-10

Focusing conditions:
Step 01    500V   Ramp: R    Time: 02:00
Step 02   1000V   Ramp: L    Time: 06:00
Step 03  10000V   Ramp: L    Time: 03:00
Step 04  10000V   Ramp: R   Vhour: 50000

total kVhrs: 71

max 50 µA
reached 50 µA limit for first 100 min, then dropped to 15µA, when voltage increased to 8000 V, 50 µA limit was reached again and dropped down to less than 50 µA only after 3 hrs (at then 10000 V).


Sjouke Hoving

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Re: 7-11 gels (Vinzenz)
« Reply #9 on: August 30, 2004, 05:00:00 PM »
Vinzenz,
The second gel looks quite good in my opionion. About the fact that you don't see many proteins between 7 and 8.5 I would not bother too much. Maybe they are not there at all!
Perhaps for rehydration you could decrease the CHAPS concentration, I don't know if it will help. I think your focusing time is sufficient. I will try this gradient once and let you know the result.

Sjouke


Vinzenz

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Re: 7-11 gels (sjouke)
« Reply #10 on: August 30, 2004, 05:00:00 PM »
Thanks Sjouke. But this is total cell lysate. And I am quite sure, that there are proteins in this range.
I like this homepage:
http://www.jvirgel.de/JVirGel.jsp?organism=Escherichia+coli+K12&Start=HTML&x=31&y=19" TARGET="_blank">http://www.jvirgel.de/JVirGel....&y=19
You can see what kind of distribution you would expect. And this is expected not to change much for mammals.
Also I did 6-9 strips and got good focusing in the range of 7-8.

Sjouke Hoving

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Re: 7-11 gels (Vinzenz)
« Reply #11 on: August 30, 2004, 05:00:00 PM »
You are right... but apparently the virtual world is different than the real world.
Have you tried a IPG 6-11, usually we get quite good results there (better than on the IPG 6-9). Could you attach an image of your 6-9 gel?

Sjouke


Vinzenz

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Re: 7-11 gels (sjouke)
« Reply #12 on: August 30, 2004, 05:00:00 PM »
Well, this is my 6-9 experience. When I used 6-11 the area of 9-11 was not focused. Now I hoped to get the whole range with 7-11.

Sjouke Hoving

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Re: 7-11 gels (Vinzenz)
« Reply #13 on: September 06, 2004, 05:00:00 PM »
Vinzenz,
YourpH  6-9 gel looks very good in my opinion.
Do you know - or others- if it is critical to use the IPG 7-11 buffer instead of IPG 6-11 for the new IPG 7-11NL with new chemistry? I tried the gradient, but had no IPG 7-11 buffer, instead I used the IPG 6-11 buffer, but the result is not satisfying.

Sjouke


Sjouke Hoving

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Re: 7-11 gels (Vinzenz)
« Reply #14 on: September 07, 2004, 05:00:00 PM »
Hi,
here is our result of an IPG 7-11NL. The sample was applied by anodic cuploading and run on a MultiPhor for about 80 kVh. But as you can see the result is not very good. Too much smearing. We used the protocol mentioned here above...

Sjouke