gel running problems - lanes are smeared lines on the gel (May/03/2005 )

Hi,
I am running a 12% gel and am having problems with my lanes being smears and I'm not seeing bands really at all (maybe some faint ones) after coommassie staining. My samples cannot be degraded because they have worked in the past and freeze-thaw is not an issue. I ran the gel at 75V for 3 hours and transferred at 30V overnight. Any suggestions?

Cat

-sophiamariapickle-

QUOTE (sophiamariapickle @ May 3 2005, 12:42 PM)

Hi Cat,
Depending on the size of your protein, you may want to consider chaging the amount of acrylamide you are using (a higher acrylamide content will give you better separation of smaller proteins, a lower content will give better separation of larger proteins). That said, two other things to consider are how long your samples sit on ice prior to loading and deterioration of the lysis buffer during long-term cold storage. Try preparing a sample with a known content of your target protein and loading it along side your samples. This way you can determine if it is a procedural error or if something has happened to your samples. I hope this was helpful and good luck!

~Sean

-Seraphi-

Hi,
Smearing on an SDS-PAGE usually means either you have too much salt in your sample and or you have DNA. For the first scenario, I would suggest that you dealt your protein prep before applying on the gel, or fo the second issue includ DNase I to your sample prep preifly (do not exceed two minutes) or sonicate your sample for shreeding the DNA in the sample. I hope this information is of help to you. Good Luck!

-bassamfahmawi-

hi,

did u denature your sample properly before loading into the gel? this happened to me before last time when i omitted this step as my lysis buffer contained urea which shouldn't be heated. after that i changed the lysis buffer and boiled the sample, the bands turned out to be very nice and no more smear.

-maymay413-