Hello everyone,
I am isolating adhesion complexes and wanted to confirm the presence of a specific protein in the isolated complex. When I performed western bott, I got a band at the right size and two more bands at lower sizes. I am using a monoclonal antibody from Abcam. I doubt that this is due to unspecific binding. Is it possible that my protein of interest was degraded? and also is it possible that is not degraded but some of them had different charge and ran faster? The predicted size of my protein is 200KDa. I used 6% SDS-PAGE. Ran for 30 min at 80V, 1 hour at 100V, and finally 120 for 45min. Do you think if I change these setup I may get better results? I have attached the pic. of the WB.
Any comment is much appreciated.
Thanks
This is quite a common occurrence even with highly specific antibodies, besides nonspecific antibody binding, other factors might the reason for instance alternative splicing, protein-receptor binding, dimerization, heterodimerization, etc. As long as you can see the meaningful differences at the correct size you can make your project conclusions. It is difficult to definitively identify what are other bands might be. With dimerization, you usually have a pattern of the double or triple size of the monomer. But more often than not these bands' exact origin remains a mystery. If you want to confirm what exactly is in the band you can excise the bands and send them for mass spectrometry analysis.
Hi there,
What kind of protocol do you use to purify the complex? Immunoprecipitation? What kind of detection do you use? Primary and secondary antibodies? If you have used your specific antibody for immunoprecipitation and the same for detection on WB then the secondary antibody will also react with the heavy chain and light chain of of the antibody used in immunoprecipitation giving 2 bands around 50 and 25kDa.
Thank you Anton Lennikov! I ran again the same sample, but this time using 8% SDS-PAGE. I also had some controls. The first lane is my positive control (the whole cell lysate of my epithelial cells), the second lane is my negative control ( the whole cell lysate of fibroblast cells as they do not express the protein of interest), the third lane is my actual samples (i.e. the isolated Adhision complexes using Ligand-coated magnetic beads). All in reducing condition. I loaded another ladder in the middle of the gel and ran my actual sample in non-reducing condition. As clear, I got one band from the whole-cell lysate (my positive control), my negative control did not show any band (as expected). However, this time I had only two bands almost close to each other (200 and 180 KDa). In the non-reduced condition, I had one band at 200 KDa (predicted size) and another one in the stacking gel that did not go through the gel. Comparing both gels, I have a feeling that something happens during the sample preparation that resulted in this outcome. Normally when I incubate my beads with the cells, I add a cytoskeleton extraction buffer to gently lyse the cells and isolate adhesion proteins attached to the beads. After removing the cell debris from the beads, I elute the beads using Laemmli buffer (boiling at 100 degrees C for 10min). I then separate the supernatant (containing the adhesion complexes) and run it on the gel. I have attached the picture of WB. I would again appreciate if you have any comment on this.
Regards,
Shahrzad
Thank you Dominique Liger! for the input.
I do not coat my beads with any antibody. I coat them with ECM proteins to isolate adhesion complexes. Briefly, I incubate epithelial cells with coated beads for one hour then I use a mild CSKB buffer to lyse the cells and then I separate the beads (that technically should carry adhesion complexes with them) using a magnetic separator. I then elute the adhesion proteins and run the sample on the gel.
You mentioned a point that I was not aware of. Although does not apply to this experiment, it is an interesting observation to consider as I have a plan to do IP as well.
Thanks again,
Shahrzad
Thanks, Ankit Pal !
Looking at the second WB I uploaded, what do you think might be the reason I do not have different variants in the whole cell lysate but in the adhesion complex. I explained my protocol in my answer to Anton. All the second bands are smaller than the first lane that appeared at the right size. Besides the possibility that post-processing of the sample might have such an outcome, is it possible that the protein-coated beads imply any changes in the expression of cell surface receptors like integrins? Can a cell line express different isoforms of an integrin in different conditions? I am asking this because for making the whole cell lysate (the fist lane from left), the cells are culture in a culture plate (unspecific binding), whereas when I use beads, the cells are technically bound specifically to a specific ligand. Do you think that could be possible?
Many thanks again,
Shahrzad
Shahrzad Nouri I don't think there is an issue with your target. Many proteins are present as double bands of the close size, especially in reduced conditions. In the non-reducing conditions you actually also see two bands the second band is barely moved at all from the top of the gel suggestive more complex secondary/triterary structure preventing its migration on the gel. Depending on the scientific question you have you may simply use this data focusing on the band of the correct size if the question is "Is this protein is present here?" then you have already answered it. If you feel that the second band is potential of great significance you can study it further by staining the gel or membrane with whole protein staining and subjecting the excised band to the mass spectrometry. You can also study the information of your protein of interest formulating the theory why in the adhesion complex several variants of the protein present of a slightly different size. You can then test this theory by incubating protein of interest with any other protein you might believe interacting with it, or overexpressing the protein of interest and it's a target on for instance HEK cells. You can also apply 2D electrophoresis to your adhesion complex to learn more about its composition. Again without knowing more about the scientific questions of the study it is hard to talk specifics.
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