negative bands problem

[SallyG]

hello

I've been trying to obtain myosin light chains and troponin bands using a 12% SDS-Page gel & silver staining, and some negative bands are appearing ie bands that are lighter intensity than the background. The bands are appearing where I would expect them to be. Please does anybody know what might be causing this? One suggestion I've had is that it might be protein overload, but firstly I don't follow why the bands would appear whitish; and secondly, on 2 separate gels (run at different times), I obtain a dark band for troponin T (one of the myofibrillar proteins) and a white band with the same concentration (from the same eppendorf tube) on the other. Any clues would be much appreciated. Thanks.

[Tfal]

well I don't really know but I might have a hint. With very high concentration of 2nd AB (HRP IgG) one gets such negative bands instead of the usual stains for the marker...

and some more piece of advice: wash, wash and wash, before, in between and after 1st AB, blocking and 2nd AB (I do at least 3-5 washes for a total of 30 min for each step)

good luck

[Tfal]

well I don't really know but I might have a hint. With very high concentration of 2nd AB (HRP IgG) one gets such negative bands instead of the usual stains for the marker...

and some more piece of advice: wash, wash and wash, before, in between and after 1st AB, blocking and 2nd AB (I do at least 3-5 washes for a total of 30 min for each step)

good luck

[Newscience]

you could try a different stain. The VisPRO 5 minute stain is a negative stain with good feedback. It takes only 5 minutes to develop and is as sensitive as Silver Stain..... its available from CHEMBIO LTD

[wolfman]

I don't have an answer but I get the same thing with my smooth muscle alpha actin western blots and have taken some good ideas from this post!