Any good ideas as to what is causing these streak would be helpful. Thanks
Details:
Second step of a taptag purification
Bis-Tris Precast Polyacrylamide gels
Ran at 165 volts
1ul sample/20ul H2O/7ul 4x loading dye/ ~37mM DTT
heat and spin samples before loading between 4-10ul to gel
Hello Jacob!
Two possibilities come to my mind:
- "Bad" interface between stacking gel and separation gel so that your samples precipitate in this area. Gradual re-dissolving of the samples could subsequently lead to streaking. Hint: during preparation of your PAGE gels overlay the separation gel with butanol (or distilled water) to get a proper interface.
- Silver staining is very sensitive. Artifacts (dust, tiny amounts of aggregated proteins, etc) will be revealed due to the high staining sensitivity.
Avoid dust when pouring the gel and ensure that there are no aggregates in your sample when you fill them into the wells (e. g., briefly spin them and pipette carefully).
Good luck!
Hello Jacob!
Two possibilities come to my mind:
- "Bad" interface between stacking gel and separation gel so that your samples precipitate in this area. Gradual re-dissolving of the samples could subsequently lead to streaking. Hint: during preparation of your PAGE gels overlay the separation gel with butanol (or distilled water) to get a proper interface.
- Silver staining is very sensitive. Artifacts (dust, tiny amounts of aggregated proteins, etc) will be revealed due to the high staining sensitivity.
Avoid dust when pouring the gel and ensure that there are no aggregates in your sample when you fill them into the wells (e. g., briefly spin them and pipette carefully).
Good luck!
Sir,
Only 2 problem comes to my mind:
1. If the purification is not proper then separation will not be proper.
2. The other is voltage pls check the voltage for electrophoresis over heat may degrade the compound if it is protein.
It might be proteins aggregated with non protein component such as DNA or lipids. You could add 1µL of DNase S1 in your sample, before heating to see if it is reducing.
But because, we see it in the molecular weight, it might be a contamination in the stacking gel or in the layer between the 2 gels. Try to redo all your buffer. If you use an alcool to do a beautiful layer between running and stacking gels, your alcool may be hydrated. Use a new bottle.
Hi Jacob
The first thing comes to mind is un clean sample prep, too much salt contents resulting in poor separation. Try and reduce sample load and run at lower voltage to allow for better separation.
It could also be very much from the staining procedure, calibrate/prewash your gel before staining.
Good luck
Hi Jacob, since you are also seeing streaks in the MW marker lane points to a problem with your loading buffer.
DTT will react with silver and DTT aggregates might be causing this problem. You could try filtering the loading buffer using a 0.1 - 0.2 micron filter.
Hi!! All above comments given by researchers can be the reason for smear in your results. You can take their suggestion. Only thing I can add is to change the used buffer and take fresh buffer (Tris-Glycine) whenever you run your SDS-PAGE for silver staining...might help you.
Good luck.
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