Anti-HA tag antibody (HRP) (ab1188)

so I loaded at least 50 ug of total protein onto the gel. ran and wet-transferred to the PVDF membrane. then incubate with the primary Ab at 4degC overnight. The primary Ab was used at a 1:1000 dilution in 5% milk in 1X TBS. I tried 1:500 dilution also and it didn't make a difference.

the next day wash with 1X TBST 5 times. then probe with the secondary anti-rabbit HRP Ab 1:5000 dilution in 5% milk in 1X TBS for 2 hrs. then wash with 1X TBST 5 times. then image with ECL or ECL plus reagent.

lot number: 51369

thanks.

ANSWER:

Thank you for your email and patience. I have tried contacting the originator of this antibody but unfortunately have not heard back. I,m not sure why the antibody is not recognizing the HA tag in your other samples. What was your order number? If you ordered in the past 90 days, I can offer you a refund or replacement antibody.

I've purchased this antibody (ab1188) and tried a Western blot... I've tried different dilutions and buffers. However, the antibody is not recognizing my HA-tagged protein in mammalian cells, only in E. coli cells. Can I return my antibody for a refund or get a similar Ab to test? It looks like ab15842 is a similar item that might be good for me to try...

Thanks,

ANSWER:

Could we get a detailed protocol from you, please? We have some general questions, the answers to which will enable us to investigate this matter as quickly as possible.

Also, please include the lot number of ab1188 that you received (it is located on the vial) and the Abcam order number of purchase order number that was used.

Thank you, and I look forward to hearing from you.

1. Please describe the problem (high background, wrong band size, more bands, no band etc).

2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein?

3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples?

4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step?

5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary?

6. What detection method are you using?

7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered?

9. Did you apply positive and negative controls along with the samples? Please specify.

I am looking to stain for an HA-tagged wingless protein in drosophila. I will be staining whole embryos and want a primary grown in Rabbit. My secondary is an anti-rabbit Cy5. I am worried about varying concentrations between batches so I think I need an affinity purified batch. I see 3 possible products, 1 being affinitiy purified and it seems to have been ordered quite a bit. I wanted to try to find any publications using this and if you guys know of any fly labs that had success with whole embryos. Thanks.

ANSWER:

Thank you for your enquiry. All the information that we have regarding this antibody is located on the online datasheet. Unfortunately, we are not aware of any publications that feature the use of this antibody. If you have any more questions, do not hesitate to contact us again.

I would like to know if this antibody HA works also in the native gel for the normal western blots, is there any special protocol? Is there any special dilution ratio for it?

ANSWER:

There is no special protocol necessary for using our antibody in this system. As long as the HA tag is exposed, the antibody will be useful. We recommend a starting dilution of 1:1000.