Anti-PADI4 / PAD4 antibody (ab26071)

Thank you for your quick response regarding ab26071. Could you please let me know how to order this blocking peptide. Please let me know the following items. 1) catalog number 2) list price (USD)

ANSWER:

Thank you for getting back to me.

The catalogue code for our PADI4 / PAD4 peptide (2-14) is ab30475. The price in USD is 150 for 100ug.

I am seeing no signal in Western blot still, even with the suggestions made over the phone. Do you have details regarding the Western blot image on your datasheet?

ANSWER:

Thank you for your enquiry.

A band at approximately 74kDa was observed in Human Spleen and Human Liver lysates (calculated MW of 74.1kDa according to NP_036519). Recommended concentration: 0.3-2µg/ml.

Below please find additional protocol details used for the gel on the datasheet. I hope this information helps. Please do not hesitate to contact us if you need anything further.

- Tissue Lysis. Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS).

- SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes.

- Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to Millipore PVDF membrane and stained with Ponceau Red to evaluate the transfer.

- Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used anti-goat-HRP for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus (Amersham) was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film.