Dear all,
I am performing Western Blots on primary HUVEC cell lysates, derived from cell cultures with fibronectin coated plates and Lonza EBM-Plus medium. I used RIPA buffer for lysis.
I performed a Western with two different antibodies. First 4 lanes are for a protein with predicted weight at 72 kD (appears to show some small bands in the 2nd and 4th lane). Last 4 lanes are for a protein predicted at 140 kD which shows bands in all lanes.
As you can clearly see there is a very intense band around 50 kD on all samples. Does anyone know what these band are, or how to ged rid of these bands? My first step will be to increase the antibody concentration, hopefully the aspecific bands will fade a little, but any input to improve the quality of this Western will be very helpful.
Many thanks in advance!
Joost
First of all the outcome of your western blot analyses is affected by various points. a) choice of lysis buffer (RIPA vs CMV or what else will lead to different patterns (try to use the same lysis protocoll fpr all samples and optimise your method at other points)); b) antibodies (specific or not, purchaser-dependent quality, concentration (maybe to high ín this case), choise of the primary/secondary host etc.); blocking procedure (BSA vs dry milk etc.), method in general (separation (e.g. time, valtage etc.); transfer (semy vs tank blot etc.)), the experimentator (routine) and so on.
Depending on quality/specificity of your antibody (for ref. see: Baker M., pp. 274, NATURE, VOL 521 (2015)) this could be a) the real (in vitro) molecular weight of your protein of interest, b) an artefact due to unspecific/ or the wrong antibodies, c) a degradation product or d) a protein of high expression accidentally containg the 6 peptide-sequence also choosen for the generation of your antibody.
In any case, your western blot analyses is not working. I would propse to revise the methode in detail. Also check specificity of your antibodies (e.g. via using recombinant protein (of interest). In consequence, you should use another (better working) antibody or perform a optimised Western blot protocol.
Many thanks for the comprehensive reply! I have thought of the antibody as being the problem, but this option is to my opinion ruled out by the fact that two different antibodies are used for the first and second half of the blot (where it was cut in half as you can see on the picture), while both blots show the same aspecific band around 50 kD. How can two different antibodies for 72 and 140 kD respectively show an aspecific band at the same height?
Perhaps I will start looking at the lysis buffer and blocking method first, or test these antibodies on a sample from a different source. Any suggestions are welcome!
The opposite approach would be more promising! You need to use less antibody, not more in order to keep the unspecific binding low, so just the real target protein should be able to bind. Think about association/dissociation equilibria and affinities of antibodies.
Of course, assuming that your antibody is not cross-reactive or that band is not a degradation product of the larger target protein containing the same epitope(s).
Try lowering the antibody concentration (e.g. 5x less), if the ratio of the specific and the (assumed) unspecific band is changing the lower band is more likely to be unspecific, if it stays the same it could be a fragment of the protein or your antibody is not specific.
In parts I agree. However, lower amounts of Abs will not solve unspecific binding. Testing of the antibody (e.g. with rec proteine) should lead to more reliable information.
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