Hello my dear fellows,
My Beta-actin for Western started to look a little strange. The bands look wide and kind of spread over, instead of being more narrow and compact. I am not sure what causes it.
I was wondering if anybody has encountered this problem before and how did you go about fixing it
Thanks!!
Dear Aleks
It seems that you have overloaded the well with protein. Load 5 times lesser protein. To see a good actin band you should load 40-50 microgram total protein.
Best of Luck
you can lower the primary antibody concentration and shorten the incubition time.
Good Luck!
I agree with the collegues, it seems a problem of over-loading. I mean, actin is a very abundant protein and primary antibodies work very efficiently. Thus, you have a very strong signal as a combination of the two reasons: the strange "morphology" of the band appears as a consequence of actin abundance, a sort of "blob". The ways to solve this point are to load less lysate, to further dilute primary antibody (or to reduce incubation time) and to expose the blot for a very short time.
I don't know whether this is relevant, but usually I run pre-cast gels at a maximal voltage of 200 V. Maybe you can also decrease a little the speed...
Hi, probably the 300V caused the typical "dumbbell" shape that you can see in the bands above the ones of actin (probably also the actin bands were affected but they were so overloaded that you can't see it). In general it is happen because the migration is too fast or too hot. It is better to decrease the speed as Stefano said.
For the actin (if you can't decrease the samples amount) I wash a lot the menbrane, I dilute the primary and also secondary antibodies . In this case, in particular, wash very well, you have a strong signal that can cause problem after the second incubation.
Hi Aleks, I give you my advices because I resolve my problems in that way. Onestly I think that it is better to run the gel with less protein, I don't know if you can have problem with the detection of your protein of interest...
From my experience, I loaded around 40ug of preotein from muscle tissue, I used max 120 V (hand-made gel), I analysed posphorylated proteins and I had problems if I increased the voltage. I had a strong, spread b-actin signal, worst than yours, so I run again at 100V, always 40ug, I diluted the b actin from 1:2000 to 1:4000 (1h or less) and I diluted the secondary antibody from 1:2000 to 1:4000 (max dilution suggested by datasheet, for 1h) all in 3%milk. Exposure time 30 sec-1min
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