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please find below the complaint form regarding ab17323, ab16230 and ab6115. I will send the FACS data per Fax (which number?). Product code: ab17323, ab16230, ab6115 Product description : Rat monoclonal to BCMA Rat monoclonal to TACI Goat polyclonal to rat IgG H&L F(ab)2 Fragment-FITC Batch number: 127950 (BCMA) 84851 (TACI) 130394 (secondary ab FITC) Date received: 23.08.05 Date first used: 26.8.05 Storage conditions used: 4C Problem: High background staining and unspecific binding of the secondary antibody -> same staining/signals with the secondary antibody alone compared to primary+secondary antibody (see FACS data) Did you repeat the experiment: YES Sample : Whole blood Anticoagulant used: EDTA Age of sample: less than 2 hours Lysis solution used: BD Have cells been activated or pre-treated in any way:NO STAINING PROTOCOL: Primary antibody details Final concentration: BCMA 1 ug/ml TACI 5 ug/ml Incubation time: 20 min Secondary antibody details: Dilution : 1:100 and 1:200 Incubation time: 20 min Temperatur: 4C Post staining fixation used : NO Time elapsed before analysis: < 5 min after incubation and washing Please give details of negative and/or positive controls that have been used: 2nd antibody only without primary or other antibody (FACS 1,2,3) 2nd antibody without these primary antibody but with Mons mAb-PECy5 (FACS7,8)
Asked on Nov 03 2005
I'm sorry to hear the customer is experiencing problems in FACS with some of our antibodies, it is likely that the problem is protocol related. I have received this morning a detailed FACS protocol with details of the buffers to use and fixative to use for staining with ab17323 and am still waiting for this information from the source of the anti TAC1 antibody, my apologies. The positive control recommended for endogenous detection by FACS is U266 cells. Recommended FACS protocol with ab17323 Buffers: PBS 1% Formaldehyde (FA) keep at 4°C PBS 0.5% saponin keep at 4°C PBS 50 mM NH4Cl 1.33g NH4Cl for 500ml Complete medium 0.5% saponin 3 x 105 cells/sample Protocol 1-wash cells 1X in PBS 2-incubate in 0.5 ml PBS 1%FA, 15 min at RT under agitation 3-wash 1X with PBS 4-wash 1X with PBS-NH4Cl 5-wash 1X with PBS 6-incubate with the first antibody in complete medium + 0.5% saponin for 30 min at RT. 7-wash once with 1X PBS 0.5% saponin 8-incubate with the secondary antibody in complete medium + 0.5% saponin for 30 min (or more depending on secondary specification) at RT in the dark. 9-wash once with PBS 0.5% saponin 10-wash once with PBS 11-resuspend in 0.2 ml PBS 12- analyse with FACS I hope this information helps,
Answered on Nov 07 2005