After immunoprecipitation with specific antibody and Protein G, samples were eluted with elution buffer, SDS sample buffer, and reducing agent at 70 °C for 5 minutes. Also, samples were incubated at 95 °C for 10 minutes before loading on SDS-PAGE.
The bubbles did not exist in the gel, and the replicate experiment shows the same result.
This is confusing because the left lane is a negative control, and the right lane is a positive control that should show immunoprecipitated protein.
Is it an protein aggregation?
Hi Kitae,
At first glance, it looks to me like a manufacturing defect in the gel. It is possible that the gel hadn't enough time to polymerize completely before letting the electrophoresis run. If this was a single case I recommend repeating the analysis. The result should be correct.
Protein aggregation optionally can occur during sample preparation or due to denaturation and subsequent refolding of proteins. Aggregated proteins may migrate differently on the gel, resulting in the appearance of vertical bands. This also could potentially be the cause of the observed band.
Best regards
Your problem is not likely to be aggregation, as very few protein aggregates persist after nearly boiling in SDS.
I agree with Jakub Knurek that the streak is probably a defect in the gel. It may appear in the same position in multiple gels from the same lot. If you bought the gels you might ask for replacements. If you poured them, next time work toward immaculate hygiene.
But that's only cosmetic. If I understand you, the real problem is that your negative control (lane 2), positive control (lane 4), and experimental sample (lane 3) all show the same single band. That extra band is missing from the MW ladder (lane 1), so it's not coming from the defective gel. It's likely an artifact from your IP method, possibly a blocker protein present in high concentration that isn't completely washed out.
I reckon you're not seeing additional bands because of underloading. But if you increase loading, your artifact band might get fatter. So while you try increasing the sample amount, also try improving wash stringency on your IP step. Sometimes it helps to increase salt concentration, but I suggest 0.1% Tween 20.
Hi there,
Coomassie staining might not be sensitive enough.
You may need to go to WB.
The bands you have might just be antibody HC and LC...
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