hi everybody,
i work with gel electrophoresis ( SDS-PAGE) , when i loading the samples with the sample buffer*4 in the wells of the gel, the samples get away and get out from the wells, the loading buffer stay in the well.
what are the reasons that can could be?
thank you,
Ammar
If you mix the samples well with the loading buffer, the samples will stay in the well. But I do not understand when you are saying that your samples get away but not the loading buffer. When you mix them properly, they can not be separated. And how do you know that your sample is getting away?
Is there any chance that your samples contain organic solvents like ethanol, methanol, or acetone?
hii budida
no i have the loading buffer that a load, it stay in the well.
the loading i do when the gel in the Running buffer
but when i loading samples that i mixed with "sample buffer *4 and heat to 95 oC for 3 min" all the sample get out from the well.
To help you better, I need more information. Is loading buffer and sample buffer are separate? And are you loading them separately? The standard protocol is to mix your samples with loading buffer, boil for 5 min and then load. If that does not work, there is problem with loading dye. The loading dye is called Lamellae buffer. And one more question is did you check whether the protein reached the gell? I hope that helps you.
Goodluck
hii Jorg, Budida,
the samples contain :75 micro-liter that taken From ( 95 micro-liter of Saline and 5 micro-liter of LB and 5 micro-liter of Oleic Acid and the enzyme that we work with it) and 25 micro-liter of Sample Buffer *4
thank you very much
Try to use other sample or loading buffer. I do not see any problem in your protocol.
For SDS-PAGE we use this buffer:
http://cshprotocols.cshlp.org/content/2008/12/pdb.rec11577.full?text_only=true
Easy to make and works well for us. ;)
If link doesn't work, the recipe is:
5% ß-Mercaptoethanol
0.02% Bromphenol blue
30% Glycerol
10% SDS
in 250 mM Tris @ pH 6.8
Do you think your sample is completely miscible with your sample buffer? I guess your sample might not be miscible with the sample buffer due to the presence of Oleic acid.
What about the purity of your enzyme? Is it an extracellular enzyme? Is it from a cell lysate? or Is it a purified enzyme?
The loading buffer and sample processing is fine but also check for the connectivity of your tank. Because if the samples are boiled well with the sample buffer/loading buffer, it cannot come out of the well.
Check for glycerol content in your sample buffer as glycerol helps to make the sample dense so the sample will remain at bottom of the well.
1. After boiling chill your samples on ice for 2 to 5 minutes before loading on to the wells.
2. if this does not work increase glycerol content of the loading buffer.
3. if your buffer is not miscible with your sample, try formamide or any other replacement of glycerol.
4. before running the gel along with samples, pre-run the gel for 20-30 mins without any sample. then clean the wells using the running buffer. now load samples.
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