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Our lab ordered and accepted monoclonal anti-TRPM7 antibody (#ab85016) and polyclonal antibody (#ab729). However we are not able to get ~216kDa or ~160 KD band of WB in HUVEC cells and HEK cells. Briefly, we used NP40 buffer to lyse washed cells. Lystae was centrifuged at max speed at 4 oC for 10 min. 5%-7% SDS-PAGE gel was used. Protein were tranfered to PVDF membrane via constant 100V for 1.5h and blocked by 5% milk/TBS-Tween. membrane was incubated with 1ug/ml (#ab85016) or 0.7ug/ml (#ab729) primary antibody, accordingly second antibody respectively and stained by ECL (amersham). I think there is no any problem but it didn't work. Do you have special protocols for both antibody?
Asked on Nov 14 2011
Thank you for contacting us. I have received WB protocol information for ab729, but am still waiting for more information regarding ab85016. Thank you for your continued patience in the meantime. For ab729, we have seen a 150-160kDa band in DDI, and in human brain lysates. All samples are boiled in Laemmli sample buffer before loading. The standard WB conditions are as follows: Starting material: We find enhancement of transfer of proteins on PVDF membranes compared to nitrocellulose membranes. • The membrane is blocked in 3% (w/v) skimmed milk in TBS-T for 1 hr at room temperature with agitation (can be blocked overnight at 4°C without agitation). • Primary antibody is incubated in the blocking buffer for 1 hr at room temperature with agitation. • The anti-goat secondary antibody should be used according to manufacturer's recommendations. Ideally, with minimal cross-reactivity with human and rodent serum proteins. It should be diluted in the blocking buffer and incubated for 1 hr at room temperature with agitation. • Wash with TBST three times after primary and after secondary antibody, each wash lasting for 5-10 minutes. • Use ECL-plus rather than ECL, which is more sensitive for detection. The blots are exposed to X-ray film. Tris buffered saline (TBS): 20mM Tris, pH7.4 in 150mM NaCl TBS-T: TBS with 0.05% v/v Tween-20 Important comments: After transfer on PVDF membrane, the quality of the transfer is monitored by staining in Ponceau red. Areas with visible air bubbles are marked with permanent ink and lanes where an air bubble was trapped at the position of bands of interest should be avoided for analysis. The Ponceau is removed by Tween-containing buffer. Primary incubation overnight at 4C requires much further dilution of the primary antibody. Still, non-specific background may be expected when incubating overnight. I might suggest trying to use RIPA buffer as lysis buffer for your samples, as the buffer is stronger than NP40 and will be able to keep the hydrophobic protein in solution better. Also, adding up to 0.1% SDS to the transfer buffer can help prevent that the protein precipitates in the gel during the transfer. Please also let me know the answers to my questions in my last email as this will be of help to determine the cause of the problem better. I hope this information is helpful to you. I will be in touch regarding ab85016. Please let me know if any of the suggestions are of help. And if not, please let me know your order or PO number so that we can resolve this issue to your satisfaction.
Answered on Nov 14 2011