Hi all,
I'm using the 7 cm IPG strip and IPGphor system for the first dimension of 2DE. Recently, I have purchased the 7 cm IPG strip holder for active rehydration prior to IEF. But I got (seems to be) a poorly focused 2D gel near the pH 10.
My protocol is, briefly, active rehydration in 7 cm IPGstrip (pH 3-10L) for 16 hrs and then IEF for 16000 vhr. Damp electrode pads were removed after voltage reached 1000V.
However, there are a series of spot appear vertically along the gel near pH 9 to 9.3...can't reach the edge of gel (edge of IPG strip)
Also, there are several horizontal streaks in the region near pH 10, besides the "column" of spots. I suspect that these streaks are proteins that could not be focused...which is outside the electrode.
So I would like to know why and how to solve the problem. I found that the gel length of IPG strip (7 cm) is longer than the distance between the electrode in the holder (~6.1cm). then, can the sample in the gel migrate to its pI (if it is very close to pH 3 and 10). And can the sample in soaked in the gel outside the electrode be focused to its pI??
Finally, what could be done except using the cup loading strip holder for IEF.
THanks for any opinion
0
2 replies to this topic
#2
-
- Active Members
-
- 90 posts
Enthusiast
Posted 08 April 2004 - 12:44 PM
IPGPhor capabilities break down near pH 10 probably because the strip composition assumes that target proteins might be more prevalent between pH 5.5 and 8.8. Can you try the more traditional BioRad apparatus? It is more pliable when it comes to extreme ranges.
Good luck
phdconsult
Good luck
phdconsult
#3
-
- Members
-
- 3 posts
member
Posted 31 January 2009 - 02:50 AM
Hi all!
My problems with my experiments IEF-2DE with IPGphor are more basic still, but if they also relate it is same band of PH 10.
Because my protein has pI 10.2. I am using stryps of 7cm pH 7-11.
Until the moment I did not obtain a protocol that me of this some satisfactory result.
Already I tried to make for cup-loadind in the Multiphor but I did not obtain nothing.
My last attempt was with a protocol treating the re-hydratation buffer
with 6% glicerol as they had recommended the protocols with very basic proteins as mine case.
Somebody could help me?
Somebody has experience and routines with 2D of very basic proteins?
I am thankful.
Roberta.
My problems with my experiments IEF-2DE with IPGphor are more basic still, but if they also relate it is same band of PH 10.
Because my protein has pI 10.2. I am using stryps of 7cm pH 7-11.
Until the moment I did not obtain a protocol that me of this some satisfactory result.
Already I tried to make for cup-loadind in the Multiphor but I did not obtain nothing.
My last attempt was with a protocol treating the re-hydratation buffer
with 6% glicerol as they had recommended the protocols with very basic proteins as mine case.
Somebody could help me?
Somebody has experience and routines with 2D of very basic proteins?
I am thankful.
Roberta.
Roberta Campos
