I have to perform WB on FFPE tissue. I've already done several attempts and the result is always the same. Can you help me understand why the bands are like this and not neat? Standard is fine (I didn't put it this time but it worked fine every time), so something went wrong before.
Some information:
- It's dog breast cancer tissue
- Deparaffination was done with xylol and a series of alcohols (there was a series of steps with centrifuging and vortexing involved)
- Pellet weight was 45 mg
- I put 100 microliter of buffer in each tube
- It was all pretty homogenized
- I tried different buffers: (1) 200 mM tris-HCl, pH 7.5, 200 mM NaCl, 5% SDS, 100 mM sodium citrate; (2) 200 mM tris-HCl, pH 6.8, 20% glycerol, 2% SDS
- I incubated the samples in a Thermobloc with a shaker for 20 minutes at 99°C and for 2h at 80°C at 100 rpm
- The gel ran for 30 minutes at 200 V
- Running buffer was already done from Bio-Rad (Tris-glycine-SDS): it was diluted depending on the manufacturer's instructions
- The materials (including gel and running buffer) are pretty old, but they told me that they should work anyway and that the problem is probably another one
I'm a beginner in laboratory, so you can say everthing that comes to your mind, even if it's something obvious.
Thanks for your help
Hi, from Rome ;)
Did the image is a gel with commassie? Did you not see the bands on gel?
Did the gel is a biorad precast? Did you run at 200V for only 30min because is in the running buffer protocol?
Valeria
Valeria De Arcangelis Hi, thanks for the answer! In the photo you can see just the bromophenol blue of the sample buffer (laemmli). Yes, the gel is a Biorad precast. The time is indicated in the protocol I used, but I tried different combinations (for example 1h with another voltage). The result is always the same
Hi Martina, if the reagents for sds-page is old but ok you have to consider a problem in your samples but I will test these samples in other way.
For example can coworkers in your lab or in another lab run one of your sample in theirs gel? Or can you can ask some ug of a protein extract (better with same protocols) from one of your coworker so you can run on your gel, as a control. In this two way you can see if your samples have some extraction issue or if the reagents that you use are ok. Did someone else is using the same reagents?
I don’t have exprerience with FFPE and the deparaffination part. After the deparaffinan you said that your recovered 45mg of cells pellet so you have a lot of start material and in 100ul it is really well concentrated.
Did you quantify the proteins before the run? Your photo is a little dark so I can’t see very well the gel but you didn’t see any degradation, you didn't have protein at all. It is possible a problem with coomassie coloration, did you use coomassie R or did you use coomassie G? Is it handmade or you buy the solution? Sometimes sds can interfere with coomassie and you have to wash with distilled water the gel before coomassie, but I didint' known the composition of biorad gel.
It is difficult to understand what is the problem because all the protocol is very long, if you can test your samples in other gels or use a control sample that you are sure is good on gel it can be of help.
Valeria
Valeria De Arcangelis Thanks for the suggestions, but unfortunately none of my collegues work with Western Blot and I don't have other materials for testing my reagents. Neither do I have something for quantification of proteins, so I have to wait until staining with ponceau S.
Sorry for the quality of the photo. It is taken right after the gel running: there is no staining of the protein, there's just bromophenol blue, the running marker of the sample buffer (Laemmli sample buffer from Bio-Rad). So I'm not even sure that there are proteins. My problem is about the behaviour of this marker, because it indicates that something went wrong (the "bands" of bromophenol should be neat).
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