SDS-PAGE protein aggregation and precipitation - (Oct/30/2006 )
Hi all,
I have been looking for the methods to minimize hydrophobic or membrane protein aggregation and precipitation for a while. Though not fully successful but now I am able to see band for my protein but still a lot remains at the top (black spot near the well; western blot). Moreover, results are variable on different days. Here are few of the things I found out at this forum and other sites and would like to have people's view on these (can you add something) esp to correct if there is any mistake:
Sample preparation:
1. Don't boil (RT for 15-20mins after adding sample buffer with reducing agent) or at 70C for 10 mins.
2. Dont' concentrate your protein too much or if you have to, then add glycerol (10-50% is the range given by different people). Can somebody correct it and give the optimum (tested) amount? Do we really need to add extra glycerol?.... sample buffer already has glycerol.
3. If you have frozen the sample, thaw at room temp (RT), make sure that SDS which is precipitated at lower temp is fully dissolved.
4. Hydrophobic protein need more SDS.....right? If yes how much in excess of sample buffer?
5. If problem with b-ME than try DTT as reducing agent? results vary from protein to protein. I read somewhere that people do add DTT to running buffer (upper chamber)... can somebody give information on that... like concentration of DTT used etc...? or is it really necessary?
6. Don't load too much protein.
7. Always add fresh sample buffer before loading (and leave for few minutes).... I add some after thawing my already prepared samples.
8. Run gel first at reduced voltage like 80mV for 10minutes (?) and then increase or decrease or if you can run at reduced voltage for overnight.. that better.
From Invitrogen site (make:
The SDS-PAGE (Laemmli system) has some potential problems:
1. The redox state of the gel is not well controlled. This means that reduced disulfides are more prone to reoxidation, giving rise to diminished band sharpness and transfer efficiency, particularly for cysteine-containing proteins.
2. Proteins are subject to cleavage at Asp-Pro bonds when heated in the Laemmli sample buffer. Also, reduction of disulfides is inefficient in the Laemmli sample buffer at pH 6.8. Either can lead to artifact bands or poor resolution.
3. The pH of the separating region of the gel is about 9.5 during electrophoresis. At this pH, proteins are potentially subjected to chemical modifications such as deamination and alkylation. This may affect subsequent analyses of proteins following the run.
Pls correct if there is any mistake
thanks.
Hi,
I do not quite understand all these points well, but the protein I have been working on does have the same aggregation problem upon heating. What I do is include 8M urea into 2XSDS sample buffer, and leave the sample at RT for around 30 mins. Also, make sure the sample buffer has relatively fresh BME added. That works well to me.
Good luck.
I have been looking for the methods to minimize hydrophobic or membrane protein aggregation and precipitation for a while. Though not fully successful but now I am able to see band for my protein but still a lot remains at the top (black spot near the well; western blot). Moreover, results are variable on different days. Here are few of the things I found out at this forum and other sites and would like to have people's view on these (can you add something) esp to correct if there is any mistake:
Sample preparation:
1. Don't boil (RT for 15-20mins after adding sample buffer with reducing agent) or at 70C for 10 mins.
2. Dont' concentrate your protein too much or if you have to, then add glycerol (10-50% is the range given by different people). Can somebody correct it and give the optimum (tested) amount? Do we really need to add extra glycerol?.... sample buffer already has glycerol.
3. If you have frozen the sample, thaw at room temp (RT), make sure that SDS which is precipitated at lower temp is fully dissolved.
4. Hydrophobic protein need more SDS.....right? If yes how much in excess of sample buffer?
5. If problem with b-ME than try DTT as reducing agent? results vary from protein to protein. I read somewhere that people do add DTT to running buffer (upper chamber)... can somebody give information on that... like concentration of DTT used etc...? or is it really necessary?
6. Don't load too much protein.
7. Always add fresh sample buffer before loading (and leave for few minutes).... I add some after thawing my already prepared samples.
8. Run gel first at reduced voltage like 80mV for 10minutes (?) and then increase or decrease or if you can run at reduced voltage for overnight.. that better.
From Invitrogen site (make:
The SDS-PAGE (Laemmli system) has some potential problems:
1. The redox state of the gel is not well controlled. This means that reduced disulfides are more prone to reoxidation, giving rise to diminished band sharpness and transfer efficiency, particularly for cysteine-containing proteins.
2. Proteins are subject to cleavage at Asp-Pro bonds when heated in the Laemmli sample buffer. Also, reduction of disulfides is inefficient in the Laemmli sample buffer at pH 6.8. Either can lead to artifact bands or poor resolution.
3. The pH of the separating region of the gel is about 9.5 during electrophoresis. At this pH, proteins are potentially subjected to chemical modifications such as deamination and alkylation. This may affect subsequent analyses of proteins following the run.
Pls correct if there is any mistake
thanks.