The ladder's probably fine, but if these are cell lines or mouse tissue you should bump it up a bit (assuming your 1Abs are high affinity and specificity for your target and are generally doing their job well). For smeared bands can be different explanations: 1) you load too much. They are detected by incubating the blot with Europium-labeled streptavidin and they show as light colored bands when read with the ScanLater Western Blot Detection Cartridge. Also, are you loading enough protein per well? The signal looks extremely faint and I can really only make out 2 sample lanes. If not it may indicate there is a problem in one of your buffers. ScanLater Western Blot Protein Ladder The ladder consists of seven biotinylated recombinant proteins at 10, 20, 40, 50, 80, 100 and 140 kD. The protein standard is supplied in a ready-to-use format for direct loading onto gels no need to heat, reduce, or add sample buffer prior to use. Then, I'd work backwards and make sure it's not my ECL reagent (is it fresh?), my 2Abs (is the concentration high enough to get a good signal?), my 1Abs (are these high affinity enough for my target? What happens if I double or half my usual concentration?), my transfer (did I transfer for long enough and did all my protein get transfered? I'd do a Ponceau or check that all dye left the gel in the case of visual dyes in the molecular ladder), and finally my gel running settings. SeeBlue Plus2 Pre-Stained Standard contains 10 proteins (4250 kDa): 8 blue-dyed and 2 with contrasting colors, for easy and quick evaluation of electrophoresis and western transfer efficiency. Because it's easy, I would maybe first play around with the length of exposure on the chemidoc and just make sure the issue isn't there first. Voltage during the run, if too high, can definitely lead to smearing in the high molecular weight range, band "smiling" or other artifacts. You may want to see if lowering the voltage a bit or switching running buffers makes a difference for the stacking and tightness of the ladder bands. Okay that helps a bit! Regarding the ladder, I assume it's detected via HRP? Or is it a proprietary BIO-RAD technology that allows it to be detected in some other wavelength range? IMO it's a little blurry and it could be stronger.
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