I am studying on cancer research and ı used bcl 2 antibody to understand there is apoptosis or not. I got good band ı tested with ponceu red. Finally I got this result.ı used uvp chemi doc it2 imager to preview membrane.I tried a lot of times what do you think about this result?
It looks like too much protein and too much antibody.
Please give more details about your procedure and image.
Did you apply ladder or not? How many samples in there?
How much Protein did you use? and how did you purify your protein? and what tissue is that? your gel constitution and electrophoresis machine used?
Hi Berna,
As Dr Mohammed comments, we need more details to understand the problem and think about how to help you. For example, may you show us the SDS-PAGE before blotting in order to analysis if the amount of protein loaded is adequated or to much, as it appears? The acrylamida percentage? Molecular weight of your target proteins? Previous treatments of the proteins? And finally, it is neccessary add a lane with molecular weight markers.
I agree, some more information would be helpful. However, there seem to be a somewhat too intensive signal to be generated in this experiment. Maybe the amount/concentration of antobodies should be reduced to obtain more "adequate"/reliable results.
Hi,
For just an addition to Elsayed Mohammed, María Luisa Caballero, Andreas Eisenreich suggestions, Beside of the appropriate protein loading and the usage of ladder markers agreement,
This also looks like a major background issue. If I'm not wrong, uneven dots or speckling ("starry sky") background problem can be seen.
Typical western blotting problems regarding this image could be caused by the followings;
- Antibodies bind to non-dissolved blocking reagents,
- HRP conjugate forms aggregates and precipitates on blot,
- contaminated transfer pads,
- corroded stirring bar or cheap skim milk (in combination with rabbit secondary antibody)
- Also, take into consideration the correct antibody concentration to get rid of glowing bands
I hope these help,
Good luck.
You really do need to annotate your image and indicate what lanes are you controls – positive and negative.
This appears to that you are imaging the dye in the sample buffer; this UV fluorescence is heterogeneous. There is nothing specific here !
I agree with everyone who suggested above that they need more information about your steps.
As u said, you got a good band with ponceau red, so everything was fine till the transfer the blot. So from this point, I can say there is a problem with your blocking steps.
If that not blocked properly, then in the next steps of probing you won't get proper bands.
Thank you so much for your interest ☺ Your right ı must to tell you more information about my protocol. Firstly, I used 2 different cell 231 and mcf 7 cell lines. And after lysis i loaded to wells 35 ul of my samples(40ug) according to this order
ladder nt vehicle ıc50. and ı used %10Res. %4 stac. gels.I used biorad transfer machine. And ı blocked with %5 milk in tbstween 25 minutes.Then ı used cell signaling bcl 2 1:1000 and Anti-rabbit seconder 1:2000.After that i got this result.
My opinion,
The specificity of the antibody on a Western blot depends largely on the sample of interest. I think we overestimate the Western blot signal because Western blotting is not a method of identifying molecules. It is necessary to prepare a positive control and negative control in the experimental system used. I also believe that it is essential to use multiple antibodies that exhibit at least independent epitopes. Please see my recent paper discussion: https://doi.org/10.1042/BSR20194012.
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