Anti-MKP-1抗体(ab1351)

We recommend using a permeablization step with this product in IHC-P. Permeabilize with 0.25% Triton X-100 for 10 minutes at room temperature. Perform this step after antigen retrieval and before blocking.

Thank you for your enquiry. I apologize for the delay in answering your question due to the holidays. The protocol used to test this antibody is below. 35ug protein per lane was loaded and its concentration would have been 2.5mg/ml in SDS Page loading buffer. The cells were not stimulated. - Lysis. Cell pellets were washed with ice-cold PBS. 1 ml of RIPA buffer was added per 1E8 cells and incubated on ice for 20 min, vortexing 2-3 times, briefly. The 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 then 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.

Thank you for your enquiry. The Western on the datasheet is detecting endogenous DUSP1 in Hela. In order to further troubleshoot why this antibody is not giving you any signal, it would be helpful if you could fill out the form at the link I am including below and submit the form. This will give us further details regarding the protocol you are using. The form can be found at: https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=1351&mode=questionaire Please do not hesitate to contact us if you need anything further.

Thank you for your enquiry and for your patience. The originator of this antibody has informed me that they only observed a single band in HeLa lysate and we have not had any other feedback yet regarding the use of ab1351. However, looking at the sequences it is possible that ab1351 could recognize DUSP4. This has not been tested. If you have any more questions, please do contact us again.