Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Converging lanes in Western


  • Please log in to reply
8 replies to this topic

#1 s056870

s056870

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 18 May 2011 - 03:06 AM

Dear all,

I wonder if anybody has seen this phenomenon.
Attached File  images for bioforum.bmp   831.85KB   282 downloads
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

#2 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 18 May 2011 - 05:20 AM

Dear all,

I wonder if anybody has seen this phenomenon.
Attached File  images for bioforum.bmp   831.85KB   282 downloads
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

#3 s056870

s056870

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 18 May 2011 - 06:48 AM

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



Dear all,

I wonder if anybody has seen this phenomenon.
Attached File  images for bioforum.bmp   831.85KB   282 downloads
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



#4 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 18 May 2011 - 09:45 PM

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




Dear all,

I wonder if anybody has seen this phenomenon.
Attached File  images for bioforum.bmp   831.85KB   282 downloads
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



#5 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
135
Excellent

Posted 19 May 2011 - 07:23 AM

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).
talent does what it can
genius does what it must
i do what i get paid to do

#6 s056870

s056870

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 23 May 2011 - 02:14 AM

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

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).



#7 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
135
Excellent

Posted 23 May 2011 - 01:15 PM

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?
talent does what it can
genius does what it must
i do what i get paid to do

#8 s056870

s056870

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 26 May 2011 - 01:30 PM

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


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?



#9 ElHo

ElHo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 90 posts
1
Neutral

Posted 06 June 2011 - 05:42 AM

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.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.