Converging lanes in Western - (May/18/2011 )
Dear all,
I wonder if anybody has seen this phenomenon.
Some of the lanes in the gels converged to become very narrow when I ran the SDS-PAGE. The bands detected were then very small. It also seemed that the bands in the narrow lanes shifted a bit upwards than the more normal lanes. Could somebody please suggest any reasons for this weird result?
Steph
s056870 on Wed May 18 11:06:49 2011 said:
Dear all,
I wonder if anybody has seen this phenomenon.
Some of the lanes in the gels converged to become very narrow when I ran the SDS-PAGE. The bands detected were then very small. It also seemed that the bands in the narrow lanes shifted a bit upwards than the more normal lanes. Could somebody please suggest any reasons for this weird result?
Steph
Hola, for me the wide bands are due to an salt excess, as the used buffer for wide bands has more ionic strength than the narrow´s. An example, if you analized the elution profile of an ion exchanger the bands width is increasing as the salt concentration arises. Have the your samples different ionic strength?
Buena suerte
Hi Buena,
Thanks for your reply.
As I understand, you seem to regard the wider bands as abnormal rather than the narrower ones, is that correct? But the thing is the wide bands have the same width as the wells for protein loading, while the narrow bands are resulted from converging lanes when the gel was being run. So, I'm not sure if I agree with your ideas.
Regarding ionic strength of the samples, I'm afraid I've never checked that. But all the protein samples were extracted from Caco-2 cells.
Thanks again for your comments.
Regards,
Stephanie
protolder on Wed May 18 13:20:23 2011 said:
s056870 on Wed May 18 11:06:49 2011 said:
Dear all,
I wonder if anybody has seen this phenomenon.
Some of the lanes in the gels converged to become very narrow when I ran the SDS-PAGE. The bands detected were then very small. It also seemed that the bands in the narrow lanes shifted a bit upwards than the more normal lanes. Could somebody please suggest any reasons for this weird result?
Steph
Hola, for me the wide bands are due to an salt excess, as the used buffer for wide bands has more ionic strength than the narrow´s. An example, if you analized the elution profile of an ion exchanger the bands width is increasing as the salt concentration arises. Have the your samples different ionic strength?
Buena suerte
s056870 on Wed May 18 14:48:07 2011 said:
Hi Buena,
Thanks for your reply.
As I understand, you seem to regard the wider bands as abnormal rather than the narrower ones, is that correct? But the thing is the wide bands have the same width as the wells for protein loading, while the narrow bands are resulted from converging lanes when the gel was being run. So, I'm not sure if I agree with your ideas.
Regarding ionic strength of the samples, I'm afraid I've never checked that. But all the protein samples were extracted from Caco-2 cells.
Thanks again for your comments.
Regards,
Stephanie
Hola Stephanie looking markers it seems that the abnormal are the nerrow ones. So,if both type of samples are extracted with the same buffer , I´m sorry I haven´t any explanation. Wait for the criterium of any of the experts of this forum. Buena suerte
protolder on Wed May 18 13:20:23 2011 said:
s056870 on Wed May 18 11:06:49 2011 said:
Dear all,
I wonder if anybody has seen this phenomenon.
Some of the lanes in the gels converged to become very narrow when I ran the SDS-PAGE. The bands detected were then very small. It also seemed that the bands in the narrow lanes shifted a bit upwards than the more normal lanes. Could somebody please suggest any reasons for this weird result?
Steph
Hola, for me the wide bands are due to an salt excess, as the used buffer for wide bands has more ionic strength than the narrow´s. An example, if you analized the elution profile of an ion exchanger the bands width is increasing as the salt concentration arises. Have the your samples different ionic strength?
Buena suerte
were they all extracted using the same buffer? detergents?
the effect you are seeing can be caused (not exclusively) by detergents or other additives to the buffer or lipids (from the sample).
Hi mdfenko,
Thanks for your reply.
The proteins from two different treatment groups were extracted using exactly the same protocol and reagents. Do you reckon it might be the treatment that made the difference? However, the problem occurred in both groups, although the majority is in one of them.
P.S. The lanes on the films are (from the left) group 1, 2, 1, 2, 1, 2, 1, 2.
Thanks,
Stephanie
mdfenko on Thu May 19 15:23:07 2011 said:
were they all extracted using the same buffer? detergents?
the effect you are seeing can be caused (not exclusively) by detergents or other additives to the buffer or lipids (from the sample).
s056870 on Mon May 23 10:14:44 2011 said:
The proteins from two different treatment groups were extracted using exactly the same protocol and reagents. Do you reckon it might be the treatment that made the difference? However, the problem occurred in both groups, although the majority is in one of them.
it depends on the treatment. what was the treatment?
also, were both groups treated equally or was there a difference in treatment?
Hi mdfenko,
Thank you for the reply.
Group 2 was treated with a flavonoid-rich food extract, while group 1 was a control in which the cells were incubated in cell culture medium only for the same period of time.
Regards,
Stephanie
mdfenko on Mon May 23 21:15:42 2011 said:
s056870 on Mon May 23 10:14:44 2011 said:
The proteins from two different treatment groups were extracted using exactly the same protocol and reagents. Do you reckon it might be the treatment that made the difference? However, the problem occurred in both groups, although the majority is in one of them.
it depends on the treatment. what was the treatment?
also, were both groups treated equally or was there a difference in treatment?
This happened to me when loading different volumes per lane. Since filling all samples to an identical final volume with extraction buffer, I've never seen that effect.