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). Also, are you loading enough protein per well? The signal looks extremely faint and I can really only make out 2 sample lanes. 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. 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. For most researchers, we generally recommend:Ī) ab116027 for proteins between 10 and 180 kDaī) ab116028 for larger proteins up to 245 kDaĬ) ab116029 for smaller proteins down to 3.5 kDaĭ) ab116029 or ab234592 for studying smaller and larger proteins at the same time.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. A protein marker (also called a protein molecular weight marker, a protein MW marker, or a protein ladder) is used to estimate the size of proteins resolved by gel electrophoresis. This mixture can include all of the proteins associated with a particular tissue. When choosing the right pre-stained protein ladder, the most important choice is based on the size of your protein of interest. A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. Prestained ladders also can use highlight bands of a different color, to make it easier to identify different size bands. Protein ladders are most convenient to use when they are supplied ready to use, with no heating, dilution, or addition of reducing agent required before loading onto the gel.
Unstained protein ladders are more accurate for sizing proteins, as the dyes used in prestained ladders can slightly distort the apparent size of the protein ladder proteins on the gel. However, an unstained protein ladder can only be visualized following staining with Coomassie or a similar non-specific protein stain. Using an unstained protein ladder is very useful when you need to accurately calculate the size of your protein. Western blot is often used in research to separate and identify proteins. Burnette in 1981 after the eponymous Southern blot for DNA and the consequent coinage of the northern blot in 1977 for RNA. calculate the approximate size of your protein by overlaying an image of the membrane with the ladder with the image generated by antibody staining. The name ‘western’ blot was first coined by Dr. 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. verify transfer efficiency between gel and membrane (PVDF, nylon, or nitrocellulose) 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. monitor protein separation during SDS-polyacrylamide gel electrophoresis Using a prestained protein ladder when running a western blot helps when you need to: