Hello to all.
I was wondering if presence of lipids can interfere with migration of proteins on polyacrylamide gels. Details are below.
I prepared an immunoliposome sample, with IgG covalently bound to some functionalized lipids. After some purification steps, I wanted to see the size and relative amounts of the IgG in several samples.
I ran them on 12% SDS-PAGE (resolving gel only), under non-reducing conditions (6X sample buffer w/o reducing agent)at 75V for 1h. After Coomassie Blue staining for 1h and o/n destaining, the bands I see are below in the picture.
Lane 1: Pre-stained protein marker. 2: Positive control, 2ug , reduced IgG (Expected size around 95-100kDa) Lanes 3-4-5: liposome+IgG column fractions
The part I don't get is, the IgG in liposome samples is supposed to be around 95-100kDa too (reduced at hinge region).But all bands are (and also in another parallel experiment)around 250kDa. I might have ran the gel for a short time, will do longer next time. But it will be great if anyone can answer the initial "lipid interference" question.
Thanks for any help in advance.
Notes:
*6X Sample buffer was prepared according to Molecular Cloning by Sambrook&Russell, I guess.
*I reviewev literature to see if people do anything different for liposome+proteins samples, could find nothing specific.
*Bands show proteins that is confirmed by spectrophotometric measurements, and same samples contains lipids also.
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asli
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Posted 20 September 2010 - 06:08 AM
asli, on 20 September 2010 - 06:05 AM, said:
Hello to all.
I was wondering if presence of lipids can interfere with migration of proteins on polyacrylamide gels. Details are below.
I prepared an immunoliposome sample, with IgG covalently bound to some functionalized lipids. After some purification steps, I wanted to see the size and relative amounts of the IgG in several samples.
I ran them on 12% SDS-PAGE (resolving gel only), under non-reducing conditions (6X sample buffer w/o reducing agent)at 75V for 1h. After Coomassie Blue staining for 1h and o/n destaining, the bands I see are below in the picture.
Lane 1: Pre-stained protein marker. 2: Positive control, 2ug , reduced IgG (Expected size around 95-100kDa) Lanes 3-4-5: liposome+IgG column fractions
The part I don't get is, the IgG in liposome samples is supposed to be around 95-100kDa too (reduced at hinge region).But all bands are (and also in another parallel experiment)around 250kDa. I might have ran the gel for a short time, will do longer next time. But it will be great if anyone can answer the initial "lipid interference" question.
Thanks for any help in advance.
Notes:
*6X Sample buffer was prepared according to Molecular Cloning by Sambrook&Russell, I guess.
*I reviewev literature to see if people do anything different for liposome+proteins samples, could find nothing specific.
*Bands show proteins that is confirmed by spectrophotometric measurements, and same samples contains lipids also.
I was wondering if presence of lipids can interfere with migration of proteins on polyacrylamide gels. Details are below.
I prepared an immunoliposome sample, with IgG covalently bound to some functionalized lipids. After some purification steps, I wanted to see the size and relative amounts of the IgG in several samples.
I ran them on 12% SDS-PAGE (resolving gel only), under non-reducing conditions (6X sample buffer w/o reducing agent)at 75V for 1h. After Coomassie Blue staining for 1h and o/n destaining, the bands I see are below in the picture.
Lane 1: Pre-stained protein marker. 2: Positive control, 2ug , reduced IgG (Expected size around 95-100kDa) Lanes 3-4-5: liposome+IgG column fractions
The part I don't get is, the IgG in liposome samples is supposed to be around 95-100kDa too (reduced at hinge region).But all bands are (and also in another parallel experiment)around 250kDa. I might have ran the gel for a short time, will do longer next time. But it will be great if anyone can answer the initial "lipid interference" question.
Thanks for any help in advance.
Notes:
*6X Sample buffer was prepared according to Molecular Cloning by Sambrook&Russell, I guess.
*I reviewev literature to see if people do anything different for liposome+proteins samples, could find nothing specific.
*Bands show proteins that is confirmed by spectrophotometric measurements, and same samples contains lipids also.
