hi, this is a 4-12% gradient gel, store bought. I added 20ug of sample to each lane, it is about 2ul volume. The sample seems to get "stuck" at the top of the gel and even though its binding to the antibody I dont see the bands.
Could this be a result of the loading dye? I had to buy new dye recently and the problem seemed to arise at that time. But could dye really do this? How do I resolve this problem?
I think this problem may come from the loading volume of your protein,on your membrane,it seems that from 150kda the samples were used out,although the amount of 20ug was no problem,but the volume of only 2ul may not enough to complete this whole running,the concentration of your protein must be extremely high,in that case I suggest you to dilute your sample equally by TNE buffer,make a enough volume of your sample(15ul/well for 15 well gel,30ul.well for 10/well gel) and try again.
I think this problem may come from the loading volume of your protein,on your membrane,it seems that from 150kda the samples were used out,although the amount of 20ug was no problem,but the volume of only 2ul may not enough to complete this whole running,the concentration of your protein must be extremely high,in that case I suggest you to dilute your sample equally by TNE buffer,make a enough volume of your sample(15ul/well for 15 well gel,30ul.well for 10/well gel) and try again.
yes try dilutions also can dilute your marker in your loading buffer and see if that impacts running
Hi Amanda,
1. What is your gel composition and what running buffer do you use? Some pre-cast gels require MOPS running buffer for example. Sometimes the loading dyes will vary depending on what running buffer you're using.
2. Is this a membrane protein? If so have you solubilized your sample using a detergent (e.g. Triton X-100)? What you've run on your gel there looks insoluble.
3. Have you boiled your sample?
4. Is there reducing agent in your loading dye? If there isn't that may be something that would help.
Good luck,
James
high concentration of protein gets clogged in gel. Dilute the protein and run.
HI, I do agree with everyone above
my suggestions
1) have you run these samples previously successfully?
2) definitely dilute your samples into at least 10µl volume. Depending on the size of your wells make sire that the volume is enough to spread at the bottom of the well, if it is too small then you end up with a blob appearance as opposed to a band.
3) Non-reducing (no DTT or mercaptoethanol in it) buffer will give you this appearance, with larger bands/smear at the top. If the dye you bought is not reducing just add for example 160 mM dithiothreitol (DTT) it works better than mercaptoethanol and less smelly! I make my own buffer as I can adjust the concentration of it i.e. for 2x concentrate of sample buffer 2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 6.8, 2 mM EDTA, 160 mM DTT and 0.1 mg/ml bromphenol blue dye. This works in any type of gels, either "home" made or pre-cast gels.
4) Are you using the right running buffer (i'm sure you do) for the right gel?
good luck
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