22 replies to this topic

[flashboy]

when i run my SDS-PAGE, it seems that themarkers don't fully separate out [there are meant to be 7 marker bands spread evenly, but only 4 appear, spread out with wide gapd between]...... does this mean that the smaller bands are not separating out? is this due to running conditions [200V for 1 hour)

cheers

[quol]

It depends on the acrylamide concentration. But genaraly the small proteins separate first. Your problem could be that the smallest proteins run out from the gel. Maybe you should run the electrophoresis shorter.

[Chris W]

Could be worth getting hold of a sample of colour markers as well to check the progress of the gel.  Could also be that the markers you have need boiling and reducing to behave as expected.

[flashboy]

is it possible that proteins will run ahead of the loading dye? if so then maybe the samples are running off the end of the gel

[phdconsult]

The gel is deficient in cross-linking.

[aj_xy999]

quol, on Jun 18 2004, 06:29 AM, said:

It depends on the acrylamide concentration. But genaraly the small proteins separate first. Your problem could be that the smallest proteins run out from the gel. Maybe you should run the electrophoresis shorter.

this most probably seems to be the case.

[zkosty]

We were having the same problem in our lab - bands running faster than the front line, very bad resolution, missing bands, additional nonexistent bands etc...
Never really figured out the real cause but the solution was simple: to replace buffers used for sds-page..
Namely, the running buffer, sample loading buffer, buffer for resolving gel, and bufer for stacking gel. A common problem is pH: These buffers are concentrated tris solutions. Such solutions are known to give wrong pH when cheap pH electrodes are used. Therefore, ALWAYS check your pH with strips - or just don't use pH electrode in this case at all.

[atul]

flashboy, on Jun 17 2004, 03:26 AM, said:

when i run my SDS-PAGE, it seems that themarkers don't fully separate out [there are meant to be 7 marker bands spread evenly, but only 4 appear, spread out with wide gapd between]...... does this mean that the smaller bands are not separating out? is this due to running conditions [200V for 1 hour)

cheers

The problem could be that very low percentage gel has been used.or else there could be coalescence of protein in marker which usually can be separated by boiling in loading buffer for 7-10 min.
                                                                       atul

[sharath]

For the marker to produce the ideal ladder, follow the manufacturer's instructions carefully. Some markers require boiling, while others do not. Some require a minute long incubation at 40C to dissolve precipitates.

Most important things, check the percentage of the gel the manufacturer used to produce the ladder. Generally 12% gel is used. I had mistakenly used 8% gel and some of  the bands didn't separate out well.

Check for expiry of the marker. I once used an old Sigma marker and many of the bands were missing.

Is your marker a premixed soultion or separate powders which you reconstitue and pool. In the latter case, be sure that all the standards are mixed in equal molar concentration ; otherwise bands of unequal intensity  ( probably missing) could result.

If you are employing silver staining then check out the manufacturer's instruction for the best staining procedure.

For best resolution you need to run the gel completely; that is, till the blue front reaches almost the bottom of the gel. Some of the bands separate out only after half the run has completed. You will notice this if you use prestained markers ( which are visible during the run itself).

Just an offhand suggestion: try running at a lower voltage ;say 80-100V

good luck

Edited by sharath, 24 February 2005 - 08:37 PM.

[ChiMy]

Could anybody suggest the reason for this case: my protein actually 30kDa but on SDS PAGE it migrate at 37kDa?

Is that the protein surface charge that involve in the migration of protein on SDS PAGE?

Thank you in advance.

[mabusheh]

ChiMy, on Mar 10 2005, 12:54 AM, said:

Could anybody suggest the reason for this case: my protein actually 30kDa but on SDS PAGE it migrate at 37kDa?

Is that the protein surface charge that involve in the migration of protein on SDS PAGE?

Thank you in advance.

Hi
On SDS PAGE, the protein migrate depending on its MW not the charge because the SDS mask the charge.  try to boil the protien in sample buffer, when I say boiling means, boiling at 100C and for at least 5 or 10  mins.

good luck

[ChiMy]

I boiled it like you say, but it still migrate strangely like that and also in some published paper they reported about this.

Some proteins also migrate unexpectedly like that, but there must be some reasons for this.
I think what I need is the characteristics of the proteins migrate strangely so that I can know more about the structure and characteristic of my protein...
Could you please suggest anything else?

[mabusheh]

Hi
ok. is this protein postranscriptionally modified, i.e, phosphorylated, glycosylated, this could change its migration pattern.
whats the gel composition, is it 10% or 12% acrylamide. current you run the gel at, or volts, all those are factors that could add to the confusion.
if you say that this is published that it runs at 37kD then whats the reason they say in this paper.

[ChiMy]

mabusheh, on Mar 15 2005, 09:01 AM, said:

Hi
ok. is this protein postranscriptionally modified, i.e, phosphorylated, glycosylated, this could change its migration pattern.
whats the gel composition, is it 10% or 12% acrylamide. current you run the gel at, or volts, all those are factors that could add to the confusion.
if you say that this is published that it runs at 37kD then whats the reason they say in this paper.

Thank you, Mabusheh!
It is a mamalian protein, phosphorylated and myristoylated but I express it in Ecoli system, so it can not get post transcriptional modification.
The gel is 15%, 100 volts for 20 min and 200 volts for 40 mins.
The paper didnt suggest anything about this, just report like that.  
Please help me figure the reason out...

[mabusheh]

ChiMy, on Mar 16 2005, 11:47 PM, said:

mabusheh, on Mar 15 2005, 09:01 AM, said:

Hi
ok. is this protein postranscriptionally modified, i.e, phosphorylated, glycosylated, this could change its migration pattern.
whats the gel composition, is it 10% or 12% acrylamide. current you run the gel at, or volts, all those are factors that could add to the confusion.
if you say that this is published that it runs at 37kD then whats the reason they say in this paper.

Thank you, Mabusheh!
It is a mamalian protein, phosphorylated and myristoylated but I express it in Ecoli system, so it can not get post transcriptional modification.
The gel is 15%, 100 volts for 20 min and 200 volts for 40 mins.
The paper didnt suggest anything about this, just report like that.  
Please help me figure the reason out...

Hi
try 10% or 12% acrylamide gels, and try to run at 80 volts for 30 min, then at 50 or 60 volts for whatever time it takes for the tracking dye to run to the bottom of your gel.
I think expressing the mammalian protein in E.Coli changes the protien somehow, isn't that right? I think the protein synthesis machinary in bacteria  adds up formyl methionine to the N terminus of the protein rather than methionine? I am not sure if there are other factors that could contribute to this. have you ever tried extracting this protein from mammalian tissue and run it on the gel and compare the differences.