Anti-DPP9 antibody (ab26177)

A while ago I wrote to you in regards to product #26177, now the customer has the following enquiry:

I'm writing with regards to an abcam product DPP9 antibody (ab26177)). I notice that there is now a picture of a western blot with antibody dilutions on the abcam site, however I would like some more information if possible. We have purchased this antibody, however have now received a replacement vial due to extremely high background signal

Therefore, I would like to know:

- What blocking buffer was used

- How long the blocking time was

- What the primary and secondary antibody was incubated with

- How long the primary and secondary incubation times were

- Whether the use of serum is necessary with any of the antibody/blocking solutions

ANSWER:

Thank you for your enquiry.

I have received the western blot procedure used to test this antibody and have copied it below. I hope this should answer your questions. However, these are only guidelines, and all individual applications may require some optimisation. I can suggest titrating to find the optimal dilution. You can try 1:1000, 1:2000 and 1:5000 to begin with. If you obtain high background, you can dilute the antibody even further.

If you have previously been obtaining high background, I can recommend trying a no-primary control (staining with secondary antibody only) in order to assess if the background is from non-specific binding of the secondary antibody. If the no-primary control gives high background, I can recommend reducing the secondary antibody concentration and incubation time. You can also try decreasing the primary antibody concentration and increasing the blocking incubation time.

Protocol for Western Blot Performed with IgY

Materials 1. Tris-buffered Saline-Tween 20 solution (TBS-T): 1.21 g Tris-base (10 mM), 8.77 g NaCl (150 mM), in 1 L of H20, pH 7.4, containing 0.05% (v/v) Tween-20. 2. Non-fat dry milk. 3. Immunoblotting blocker reagent. 4. HRP-conjugated anti-IgY Fc antibody. 5. Immuno-blot colorimetric assay kit.

Method 1. Separate appropriate amount of cell lysates (20-30ug protein per lane) by 10-20% SDS-PAGE, followed by transferring to PVDF membrane. 2. Block membrane with 5% non-fat milk in TBS-T (Tris-buffered saline containing 0.05% Tween, pH 7.4) for 1 hour at room temperature or overnight at 4oC. (BSA is not recommended as a blocking reagent). 3. Rinse membrane with TBS-T. 4. Incubate membrane with ab26177 appropriately diluted with 5% milk in TBS-T @ R.T. for 1 h. Option: pre-incubate diluted IgY with Immunoblotting blocker @ R.T. for 1 h prior to submerging membrane. This step may help to reduce background, especially when E. coli-derived antigen is used. 5. Wash membrane with TBS-T, 3 min each, total of 3 times. 6. Incubate with 2nd antibody (anti-IgY/Fc-HRP) with 0.5% milk TBS-T) @ R.T. for 1 h. 7. Wash with TBS-T, 3-5 min each with shaking, total of 3 times. 8. Perform color development. Or perform ECL detection of signal using ECL kit.

I hope this information is helpful. Please do not hesitate to contact me again should you have any more questions. Good luck with your research!

BATCH NUMBER 171136 ORDER NUMBER 19031

DESCRIPTION OF THE PROBLEM High background - film is completely black after a 10 second exposure

SAMPLE DPP9 transfected 293-T cells

PRIMARY ANTIBODY Chicken polyclonal to DPP9 (Abcam, Cat #ab26177).

Tried concentrations of 1/500, 1/1000 and 1/2000 in blotto, with and without horse serum. Incubation time = 1.5hrs.

Washing = 3 x 8 minutes in blotto.

DETECTION METHOD SuperSignal West Pico Chemiluminescent Substrate (Pierce, Cat # 34080)

POSITIVE AND NEGATIVE CONTROLS USED Secondary antibody only (no primary added to membrane)

ANTIBODY STORAGE CONDITIONS -80 degrees celcius (in 5ul aliquots)

SAMPLE PREPARATION PBS with protease inhibitor cocktail. Sample is boiled (100 degrees celcuis for 10 minutes) in 6X SDS buffer prior to loading on a gel.

AMOUNT OF PROTEIN LOADED 20ug

ELECTROPHORESIS/GEL CONDITIONS 7.5% acrylamide gel (non-reducing).

TRANSFER AND BLOCKING CONDITIONS Transfer to PVDF membrane for 1.5hrs at 60V in SDS transfer buffer (25mM tris, 152mM glycine, pH 8.3)

Membrane is blocked in blotto (PBS; 0.05% tween-20; 5% w/v skim milk powder) overnight at 4 degrees celcius.

SECONDARY ANTIBODY Peroxidase conjugated affinity purified anti-Chicken IgG [H&L] [Goat] (label = HRP) (Rockland, cat # 603-103-002)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Concentrations of primary and secondary antibodies, incubation of primary antibody in the presence of horse serum.

ANSWER:

I'm sorry to hear you are experiencing problems with ab26177.

The problem you are experiencing is likely to be due to either the antibody or the blocking buffer as well as the fact that you are running a non-reducing gel (denaturing and reducing conditions are recommended). I would recommend to try the following blocking methods: -try blocking the membrane in 5% BSA in TBST for 1 hr at room temperature, then rinsing lightly the membrane in TBST and incubating the membrane in primary antibody in TBST. Use also TBST only to dilute the secondary antibody and for washes. Too much blocking agent can cause high backgrounds too unfortunately. -try also the same with 5% milk.

You use a very performant detection system and I would like to suggest to dilute the antibody much more. It is best to add the antibody very diluted and incubate it overnight, as this promotes a slow but targeted binding to the specific protein of interest. You may therefore be able to dilute to 1:5000 or more. This antibody has only been tested against recombinant fusion protein therefore I'm afraid I cannot recommend a good positive control which will have high levels of the protein and clarify whether you have a good expression of the protein in your transfected cells.

Finally I would recommend short multiple washes in TBST (6 washes of 5 min each) to make sure any excess antibody is washed away.

I hope the above recommendations will help you. If you still experience problems with those changes please do not hesitate to contact me again,