Anti-NAIP antibody (ab25968)

First of all, I find it exceedingly difficult to ask a simple question regarding products that have been ordered from your compnay and paid for already (one of these not yet been received). Anyway, my question is rather simple, I have purchased the antibody ab25968 as well as the associated peptide (this is what I am waiting for). In the datasheet, you specify that a good positive control for this antibody is the PC-3 cell line. A) What is the secondary antibody used? and B) How was the PC-3 lysate prepared? I have used antibodies developed by other companies and observed a lot of non-specific binding. My desire is to mimic your experiments as close as possible so that I can observe something similar to what is shown on the datasheet. Is the lysate prepared by the NP-40 method or something different. Thank you.

ANSWER:

Thank you for your enquiry.

I have enquired at the originator of the product for the WB protocols. Please see below for further details. I hope this information will be helpful.

To answer your questions:

1. We us an anti-rabbit IgG, HRP conjugate.

2. We use a modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin). After centrifugation and determining the protein concentration, the lysate is boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.

Below are the protocols that we use.

Buffer Formulations:

SDS/Running Buffer: • 25 mM Tris • 192 mM Glycine • 0.1% SDS

Transfer Buffer: • 20 mM Tris • 150 mM Glycine • 20% methanol 0.038% SDS

Blocking Buffer • 5% non-fat dry milk • TBS

Wash Buffer (TBST) • 125 mM NaCl • 25 mM Tris pH 8.0 • 0.1% Tween-20

Running Protein Samples onto a Gel

1. Cast a mini SDS-PAGE gel per your labs standard protocols or purchase pre-made gels.

2. Clamp the gel to the apparatus with per manufacturer directions. Add running buffer.

3. Remove the comb gently so as to not disturb the wells.

4. Add 6μL of your select marker to a well.

5. Add 7.5μL of lysate per well (2mg/mL for 15μg per lane).

6. Apply the anode and cathode wires to the appropriate poles and cover.

7. Run at 130V for 2 hours (or until the dye front is close to the bottom).

Transfer of Proteins onto Membrane

8. Assemble the “sandwich” for the transfer apparatus per the diagram below.

a. Note: Handle gel and membrane with tweezers – do not touch!

9. Pre-wet sponges and filter paper in transfer buffer.

a. Note: Filter papers and membrane should be same size as the gel.

10. Insert the “sandwich” and insert into the transfer apparatus, making sure the gel is on the cathode (-) and the membrane is on the anode (+) side of the apparatus.

11. Transfer the proteins to the membrane at 250mA in transfer buffer for 2 hours

12. Remove the cassette from the apparatus, discard the gel, and place membrane on paper towels to dry. (Wash the membrane in 1xTBST to remove any leftover gel.)

Blocking

13. Incubate the blot with blocking buffer overnight at 4°C or 2 hours at room temperature with gentle agitation.

14. Remove blot from blocking solution.

Primary Antibody Incubation

15. Dilute antibody to the recommended dilution in 10mL of blocking buffer. 16. Incubate the blot with the primary antibody for one hour at room temperature or overnight at 4°C.

17. Wash the blot three (3) times 10 minutes each in washing buffer with gentle agitation.

Secondary Antibody Incubation

18. Dilute 1μL anti-rabbit IgG-HRP conjugated secondary (or other appropriate secondary) in 10mL of blocking buffer to make a 1:10000 dilution

Note: working dilution of secondary can vary from 1:2000 to 1:10000.

19. Incubate blot with secondary antibody for one (1) hour at room temperature.

20. Wash three (3) times for 10 minutes each in washing buffer with gentle agitation.

Development

21. Drain wash buffer

22. Add ECL solution (Amersham) per manufacturer directions and develop for 1 minute.

23. Drain the fluid.

24. Cover the blot in plastic wrap.

25. Expose the blot to X-ray film for 1 minute in a dark room.

26. Develop the film in an X-ray processor

Note:

a. If there is no banding, expose the film for 5 minutes, then 30 minutes and up to overnight if the signal is weak.

b. If the signal is strong, expose the film for 30 seconds or less.

c. Optimal dilutions should be determined by each laboratory for each antibody