Hi researchers,
I have been running a western blot setup optimisation with beta actin as a loading control protein. I am getting non-specific bands at the higher MW range in addition to my target beta-actin band.
These were my key steps:
1. Gel was made at 16.5% (as I was probing a smaller MW protein at 5kDa as well) and samples were run at 120V for 90 mins.
2. Transfer of gel was done at 10mA overnight in the cold room (4 degrees celsius).
3. Transfer done as per bio rad protocol, and following remove of PVDF membrane, blocking was done in 5% BSA in TBS-T for 1 hour and r.t., following which anti-beta actin was added in a 1:10000 ratio (antibody from protein tech, mouse monoclonal). But primary antibody was in 1% BSA in TBS-T.
4. Pri incubation was overnight in cold room as well.
5. Next day, after 3-5 washes in TBS-T (each was 5 minutes), secondary incubation with 1:20000 of anti-mouse antibody (diluted in 1% BSA in TBS-T as well.
Is it something to do with antibody dilution? I have read article that used even more dilute concentrations for beta actin (1:100,000 for eg.), and I am not sure that the higher concentration could have resulted in the extra bands. Or the fact that I incubated my antibodies in 1% BSA, as opposed to maintaining the BSA % used for blocking.
Kindly refer to the attached image for the blot.
If someone has experience in this aspect, it would be great to hear from you!
Thanks and Regards,
Mathangi
Hi Mathangi Palanivel
You may try using a more dilute concentration of beta actin antibody. But it may not help. I make the primary and secondary antibody dilutions in 3% BSA in TBST and blocking is done in 5%BSA in TBST. The rest of the protocol seems okay to me.
Besides the above, I would like you to know that although actin is often thought of as a single protein, in mammals, it consists of six different isoforms encoded by separate genes. Each isoform is remarkably similar to every other isoform, with only slight variations in amino acid sequence. So, there is a high possibility that the beta-actin antibody which you are using may not be specific.
You may try the most cited anti-beta-actin antibody clone AC-15, which is used in Western Blot. AC-15 belongs to the subclass IgG1 and was raised in mice against the N-terminal synthetic peptide of beta-actin. The clone specifically targets the beta isoform, with no cross-reactivity against the other 5 isoforms of actin family.
I have attached a link below regarding the anti-beta-actin antibody clone AC-15 for your reference, which may help.
https://www.abeomics.com/monoclonal-antibody-to-anti-beta-actin-antibody-clone-ac-15
Best.
Malcolm Nobre ok thank you! Will look into it. I also did a stripping of the membrabe before subjecting it to invubation with beta actin antibody. the protocol was to incubate 45 mins in 50 deg, shaking with stripping buffer containing beta mercaptoethanol, before washing with running water for 1 hour. Not sure if stripping was not done adequately as well.
I'm not sure if this is the antibody you have (https://www.ptglab.com/products/Pan-Actin-Antibody-66009-1-Ig.htm) but if it is, the website recommends a dilution range of 1:20,000 - 1:100,000. I personally use 1:20,000 (albeit with a different beta-actin antibody) and I get really good signal without non-specific bands.
Non-specific bands can appear when your antibody is not diluted enough.
Carles Puig Saenz ok thank you! Yup thus is the Ab im using and I used 1:20,000 as well. But I could try a more dilute concentration as I have seen some others who used 100,000 for the same Ab. May I check what is the conc of the secondary antibody that you used?
Of course, by all means try larger dilutions; if you have samples to spare, why don't you try a range? For example 1:40,000, 1:60,000, 1:80,000 and 1:100,000?
For the secondary I always use 1:1,000; but my secondaries are from Cell Signalling (https://www.cellsignal.com/products/secondary-antibodies/anti-mouse-igg-hrp-linked-antibody/7076?_requestid=714868) and they always recommend to use 1:1,000 - 1:3,000.
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