Hello everyone, I'm a master student and in my project I'm studying RFP-based constructs (RFP + few amino acid-long tags). I'm now performing a Western blot in SDS-PAGE of my cell-line lysates.
My western blots (as the photo shows) have a problem in the Ponceau staining as the marker is fairly visible and correctly transferred, however my sample lanes show little to no protein (I've loaded 50 ug), especially in the second half of the blot. The same problem is present also with other types of samples (like whole cell lysates) and seen in some of my collegues' blots.
I've tried make a new batch of sample buffer (+ B-mercaptoethanol), new acrillamide (gel is usually 10%) and new transfer protocol (10 minutes, 25V).
Another weird thing I've seen is that incubating an antibody (any kind really, I've tried B-actin), the signal is confined in the area of the blot that corresponds to where the stacking gel meets the running gel.
I thank whoever offers suggestions.
Hello Irene,
It seems that the sample has troubles to enter the running gel.
Here are my ideas:
- Dry running gel - the runnig gel dried during preparation, if you cast running and stacking gel separately, it is necessary to keep it wet by isopropanol layer during polymerization
- sample buffer is too dense - I don't know, which sample buffer you use, but it could be too dense. We use 1x Ripa or 1% SDS or their mix. We use the same buffer or water for sample dilution during preparation for WB. Also we use 4x Laemli buffer (Biorad), 900 ul of 4x L. buffer + 100 ul b-mercaptoethanol and mixing with the sample 1:4 ratio (Laemli b. to diluted sample) is recommended.
Thank you so much Helena Skálová I'm trying to change the sample buffer but it makes sense (?) that it's a problem with the running gel. I'll try adding more isopropanol as I prepare it. I also tried some Comassie blue gel staining and it shows more or less the same thing, so it's definitely one of the 2 things you suggested.
I've finally figured out the answer: the problem was in the electrophoresis equipment: the buffer was leaking and therefore the gel wasn't fully covered. I write this down in case other people have a similar problem, to check this I had to keep an eye on the mAmpere of the machinery (lower than 35 mA but higher than 20 mA).
I thank everyone who has been interested.
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