On Abcam datasheet says when using ab2090 or ab2074 there is only 1 band detected around 43kDa. They tested the antibody in HeLa whole cell lysate.
I used ab2074 in HUVEC and HMEC whole cell lysate and I always got more then 1 band ( image 1 green and red bands). I know that using rabbit polyclonal antibodies there is more unspecific bands that can be detected and because of this I ask Abcam if there were more bands that could be detected and they said no.
I did some research and I found a paper where they detected multiple CXCR4 isoforms in neuroblastoma cells, with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to null surface expression (image 2)
Carlisle, A. J, Lyttle, C. A, Carlisle, R. Y, & Maris, J. M. CXCR4 expression heteroge‐ neity in neuroblastoma cells due to ligand-independent regulation. Molecular Cancer (2009). , 8, 126-131
In the same paper they say :
“Post-translational modifications, some of which may be associated with constitutive protein turnover and agonist-induced trafficking of CXCR4, could affect the surface density and conformational forms of CXCR4 and have an impact on the overall susceptibility of a given cell for HIV-1 infection. Some previously reported modifications of CXCR4 include N-linked glycosylation and sulfation. “
I need to quantify the total protein CXCR4 and see if there are differences between HUVEC and HMEC. So, i would need to confirm that Im detecting other isoforms of CXCR4 and if so I was thinking to quantify all bands toguether and also quantify separately the bands , because they have different expression between the 2 cell lines.
I would need same feedback to know if this way is ok or if there are other ways to quantify.
Some of the bands you are seeing are probably dimers - and CXCR4 is a dimer. That's normal and you can add bands that are double of the expected size to your calculations. I would also recommend increasing Tween concentration in your Western wash buffer because you may have unspecific Ab binding as well.
Thanks Anton for your tips.
I was using 0.05% Tween 20 in TBS1x and was doing 3x-10min wash.
You can vary 0.1 - 0.5% Tween 20 in primary or secondary antibody solution and in wash buffer. Another option is to decrease concentration of primary antibody. Finally, you can load less protein in each lane.
For the primary antibody I was diluting in Block Buffer from milipore (specific for Fluorescent Detection) so dont know the concentration of Tween20 in the buffer, although I can ask them.
I was loading 12 ug of protein and I would think less could decrease the signal of the bands below 50kDa that is the area where should be the 43kDa band (CXCR4) that abcam says in the datasheet.
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