hi im new to research so i will be happy to receive any advise on this matter. I have a new cell line with me and i am proceeding to ensure that this cell line is indeed what it is by detecting protein expression of certain markers.
i extracted total protein lysate and proceed to load 50ug of total protein into SDS, followed by antibody detection with western blot. I read from many sources that 50ug is a good 'chance' of success but for 3 main proteins im detecting, i am getting faint bands and have to resort to even longer exposure time to get something decent. The thing is my housekeeping protein (actin) also have similar faint bands which led me to think that maybe protein is degraded or transfer is not complete.
2 of the proteins is 100kd, 1 protein is 50kd. and i perform transfer with 1.5mm SDS gel at 80v for 2 hours. to be honest, the protein ladder markers gets transferred ok for the 100 and 50kd regions but my ponceu stain showed that my samples dont really have strong discrete bands. more like a few bands here and there and some regions even have smear instead of discrete bands.
i could try with a different lysis buffer but what other conditions would you recommend that i tweak??
thanks in advance!
What method are you using to quantify protein? Don't use Bradford for this. If you use BCA make sure you add beta mercaptoethanol after the protein assay, or purchase the reducing agent compatible BCA kit. In all cases your samples should be on ice except for very brief cell disruption where you can briefly remove sample from ice. Centrifugation should also be in a refrigerated centrifuge. Once beta mercaptoethanol and loading dye is added then you can warm up the aliquotstairs of your samples. Insider tip here: if you are looking at membrane proteins do not boil your sample for those tubes, instead mix with loading dye and warm to 37 Center for 30 min.
Hello Iwan
As Adam said, you have to maintain your sample in ice through out. Make sure that you have added adequate protease inhibitor cocktail. You have to do protein quantification ( either Bradford or BCA method are fine).
Boil the sample at 70 degree for only 90 seconds (for membrane proteins warm the sample as Adam mentioned). 100 v for 1 hour worked better with me. you can try that.
I think your proteins is degraded and faint band of housekeeper proteins in addition to smear suggest that, just add sufficient protease inhibitor cocktail and always keep samples on ice.
Good luck
- Badges
- Science method