I am looking for an HA-tag antibody to use with yeast. The HA-tagged proteins I am trying to assay via WB are endogenously tagged with 3xHA. I don't see very high levels of expression. Therefore, I need a highly specific antibody with essentially no non-specific activity against yeast whole-cell extracts. Does anyone have a recommendation?
I've used a couple of antibodies 1) HA Tag Monoclonal Antibody (2-2.2.14) [Invitrogen 26183], shown below, and 2) Anti-HA (3F10) [Roche 12013819001]. Both have a lot of non-specific activity with non-tagged proteins that can be observed in the WT lane and both antibodies have the exact same background band pattern and intensity.
The calculated molecular weight is 97 kDa for SCH9-3HA and 81 kDa for YPK1-3HA.
Thank you for your suggestions!
Kendall C. Case , have you tried to titrate your secondary AB for low background, i.e. dilute it as much as possible?
Wolfgang Schechinger, I am already using a 1:5000 antibody dilution. I can try a 1:8000 on my next WB. My concern is the signal from my tagged protein also decreasing. I'm also considering using more protein lysate, in which case maybe a 1:10,000 dilution would be better yet. Thank you for the recommendation!
Kendall C. Case It's the signal to noise (=background) ratio that counts. Absolute intensity is for the human eye, as well as are apparent intensity ratios. When you integrate the signals (e.g. with ImageJ), could you live with the results?
As you seem to use camera based imaging, you may extend the exposure time when you are using higher dilutions. The systems usually allow you to either take time series or even to save snapshots at different integration times. Playing around with gradation scales (lin vs log etc.) also might change the optical impression of the signals you observe.
Other things you could try is to pre-incubate the antibody dilution with HA-tagged protein to find out which bands are specific and which are not.
Also playing around with the conditions for the hybridization might give "better" results. Changing salt concentrations or the ionic composition might give different results, too. Depends on how much time and effort you want to invest.
Hi, I have used several anti-HA-Tag antibodies, among them, C29F4 (CST) and 3F10, from my experience, are the best. I am sorry I only have experiences to analyze samples from mammalian cells. The affinity of 3F10 to HA is usually too high for me, I mainly use C29F4 for western-blot with both good specifity and high sensitivity. Since you are analyzing 3HA tagged endogenous protein, I think 3F10 should be very good one. To reduce the background, it might be good to incubate the membranes with the dilutued antibodies (I often use 1:1000 for both primary and secondary Abs) in some comercial Immunoreaction Enhancer Solutions (I use Can Get Signal from TOYOBO).
Thank you Yicheng Wang for your recommendations! I have tried the 3F10 antibody and it seems to have less affinity and less specificity than the 2-2.2.14 antibody I am currently using. I think it would be worth trying the C29F4 antibody (CST) you recommended.
I've been working with HA Tag Monoclonal Antibody (2-2.2.14) [Invitrogen 26183]. I was able to get slightly less background using TBS-T with 0.1% Tween rather than 0.05 %. I used a 1:5000 dilution.
For anyone who cares, @Yicheng Wang offered the best antibody alternative, 3F10, which is Anti-HA High Affinity, rat monoclonal antibody (Roche 11867423001). It has great specificity and affinity (as the name implies).
You can see the difference on the attached WB image where I assayed SCH9-3HA lysates.
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