10 replies to this topic

[Cole Ziegler]

I have uneven lane widths. I'm only using two samples, alternating every other lane.

What you will see is that the samples are not causing the lane width problems. For instance, on the first image, it is not the sample 1881 that is the skinny lane, but it is in the second image. Furthermore, the first lane is skinny on the first image, but it is the second lane that is skinny on the second image.

Hippocampus brain tissue. Bio-Rad criterion TGX MIDI gel (Any kD formula). Bio-Rad Transfer MIDI Trans-blot transfer membrane. Stained with memcode.

Thanks in advance.

[science noob]

1a. Did you load equal amounts of loading volume? (sample+loading buffer)

1b. Did you do a protein estimation to make sure equal amounts of total protein was added?

2. Were all samples loaded onto the wells at the same time?

3. What was the SDS-PAGE run setting (V? time? A?)

[Cole Ziegler]

science noob, on 29 June 2012 - 03:28 PM, said:

1a. Did you load equal amounts of loading volume? (sample+loading buffer)

1b. Did you do a protein estimation to make sure equal amounts of total protein was added?

2. Were all samples loaded onto the wells at the same time?

3. What was the SDS-PAGE run setting (V? time? A?)

1a. Yes.
1b. Yes.

2. Yes.

3. 180 V for 80 min @ 3.0 A

[bob1]

I would suspect that either the gels are a bit dodgy (how old are they? expiry date?) or the salt content of the samples is a bit funny for some reason.

[Cole Ziegler]

bob1, on 29 June 2012 - 11:24 PM, said:

I would suspect that either the gels are a bit dodgy (how old are they? expiry date?) or the salt content of the samples is a bit funny for some reason.

Gels are precast from Bio-Rad and are less than two weeks old. Shelf life is supposed to be 12 months. Expiration date on them is 05-29-2013.

That would be interesting if salt content was the issue because this same exact protocol worked fine until yesterday and today. Not ruling out the possibility of salt being the issue, it's just weird that it is coming up now.

[Papaver]

Well, sometimes I had the same "problems" but I've never worried about that. Usually it depended on my samples I loaded onto the gel especially when it was a whole extract (e.g. cytoplasm and membrane extractions).
Do you need a nice image for publication? If not and if you see everything you need to see why worring? (Sorry, if I possibly play down your problem...)

Edited by Papaver, 30 June 2012 - 05:32 AM.

[Cole Ziegler]

Papaver, on 30 June 2012 - 05:23 AM, said:

Well, sometimes I had the same "problems" but I've never worried about that. Usually it depended on my samples I loaded onto the gel especially when it was a whole extract (e.g. cytoplasm and membrane extractions).
Do you need a nice image for publication? If not and if you see everything you need to see why worring? (Sorry, if I possibly play down your problem...)

Yes, I do need a nice image for publication. Also, because of the lane differences, my memcode analysis shows that the lanes are not loaded with the same amount of protein (even though they are all loaded with 10 ul). The reason why it is not equal between all lanes is because of the width differences.

[science noob]

Cole Ziegler, on 30 June 2012 - 10:24 AM, said:

Papaver, on 30 June 2012 - 05:23 AM, said:

Well, sometimes I had the same "problems" but I've never worried about that. Usually it depended on my samples I loaded onto the gel especially when it was a whole extract (e.g. cytoplasm and membrane extractions).
Do you need a nice image for publication? If not and if you see everything you need to see why worring? (Sorry, if I possibly play down your problem...)

Yes, I do need a nice image for publication. Also, because of the lane differences, my memcode analysis shows that the lanes are not loaded with the same amount of protein (even though they are all loaded with 10 ul). The reason why it is not equal between all lanes is because of the width differences.

In this case, you can still analyse this image even if it isn't publication perfect.  The image software I use (Quantity ONE from BioRad) measures band intensity x volume (of the pixelated bands after chemiluminescent exposures.  Therefore, if you had the same sample +volume but different lanes (one wide, one narrow), you would expect to see the narrow one being more intense (protein jam-packed in that small area) vs. wider band which are more spaced out.

[Cole Ziegler]

science noob, on 30 June 2012 - 03:58 PM, said:

Cole Ziegler, on 30 June 2012 - 10:24 AM, said:

Papaver, on 30 June 2012 - 05:23 AM, said:

Well, sometimes I had the same "problems" but I've never worried about that. Usually it depended on my samples I loaded onto the gel especially when it was a whole extract (e.g. cytoplasm and membrane extractions).
Do you need a nice image for publication? If not and if you see everything you need to see why worring? (Sorry, if I possibly play down your problem...)

Yes, I do need a nice image for publication. Also, because of the lane differences, my memcode analysis shows that the lanes are not loaded with the same amount of protein (even though they are all loaded with 10 ul). The reason why it is not equal between all lanes is because of the width differences.

In this case, you can still analyse this image even if it isn't publication perfect.  The image software I use (Quantity ONE from BioRad) measures band intensity x volume (of the pixelated bands after chemiluminescent exposures.  Therefore, if you had the same sample +volume but different lanes (one wide, one narrow), you would expect to see the narrow one being more intense (protein jam-packed in that small area) vs. wider band which are more spaced out.

I agree. However what do I do when I do need a publication image? What would be causing this problem?

[mdfenko]

as bob1 pointed out, we see this effect when we have different salt concentrations in the samples.

we also see it when there is lipid in the sample.

[Cole Ziegler]

mdfenko, on 02 July 2012 - 07:47 AM, said:

as bob1 pointed out, we see this effect when we have different salt concentrations in the samples.

we also see it when there is lipid in the sample.

My salt concentration is 150 mM NaCl and is prepared for both samples (i.e. both samples are homogenized in 100 ul RIPA lysis buffer from the same solution). I don't see how the salt concentration could be different between the two samples then.

Could it be that my RIPA lysis buffer from Millipore is just about to reach its expiration date? http://www.millipore...gue/item/20-188