8 replies to this topic

[s056870]

Dear all,

I wonder if anybody has seen this phenomenon.
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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

[protolder]

s056870, on 18 May 2011 - 03:06 AM, said:

Dear all,

I wonder if anybody has seen this phenomenon.
images for bioforum.bmp
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]

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 18 May 2011 - 05:20 AM, said:

s056870, on 18 May 2011 - 03:06 AM, said:

Dear all,

I wonder if anybody has seen this phenomenon.
images for bioforum.bmp
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

[protolder]

s056870, on 18 May 2011 - 06:48 AM, 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 18 May 2011 - 05:20 AM, said:

s056870, on 18 May 2011 - 03:06 AM, said:

Dear all,

I wonder if anybody has seen this phenomenon.
images for bioforum.bmp
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

[mdfenko]

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]

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 19 May 2011 - 07:23 AM, 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).

[mdfenko]

s056870, on 23 May 2011 - 02:14 AM, 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?

[s056870]

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 23 May 2011 - 01:15 PM, said:

s056870, on 23 May 2011 - 02:14 AM, 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?

[ElHo]

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.