Source | M.W. | ~45 kDa | CAS No. | ||
---|---|---|---|---|---|
Structural Info | |||||
Formulation | Lyophilized in sterile filtered solution of PBS with 2% CHAPS | ||||
Reconstitution | Before reconstitution, werecommend a brief spin to drive down any material dislodged from the bottom ofthe tube. The lyophilized protein shouldbe reconstituted in sterile H2O to a concentration of 100 ng/uL. Because of the hydrophobic nature of thisprotein, further dilutions should be made in buffer or medium containingcarrier proteins, such as albumin or serum. | ||||
Stability | The lyophilized protein isstable for at least 1 year if stored at -80 °C. Reconstituted protein is stable for at least1 month at 4 °C, but should be stored in aliquots at -80 °C forlonger term. Avoidrepeated freeze and thaw. | ||||
Purity | Greater than 85% as determined by SDS-PAGE and HPLC analysis | ||||
Biological Activity | The activity was determined by using a TCF reporter gene assay in 293 cells co-transfected with Frizzled-4 and LRP-5.WNT-5a activates (instead of inhibits) the TCF reporter gene in this assay. | ||||
Country of Origin | USA |
WNT-5a belongs to the class of WNT proteins thatactivate the “non-canonical” pathway. Thepredicted size of mouse WNT-5a is a monomeric protein containing 356 amino acidresidues. Due to glycosylation, itmigrates at an apparent molecular weight of ~45 kDa by SDS-PAGE analysis undernon-reducing conditions. StemRD’sproduct is expressed from a mouse cell line, and purified with a proprietaryprocess that is distinct from the published method.
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目前大量制备单抗的方法主要有两大系统,一是动物体内生产法,这是国内外实验室所广泛采用;另一是体外培养法。
(1)动物体内生产单抗的方法
迄今为止,通常情况下均采用动物体内生产单抗的方法,鉴于绝大多数动物用杂交瘤均由BALB/c小鼠的骨髓瘤细胞与同品系的脾细胞融合而得,因此使用的动物当然首选BALB/c小鼠。本方法即将杂交瘤细胞接种于小鼠腹腔内,在小鼠腹腔内生长杂交瘤,并产生腹水,因而可得到大量的腹水单抗且抗体浓度很高。可见该法操作简便、经济,不过,腹水中常混有小鼠的各种杂蛋白(包括Ig),因此在很多情况下要提纯后才能使用,而且还有污染动物病毒的危险,故而最好用SPF级小鼠。
(2)体外培养生产单抗的方法
总体上讲,杂交瘤细胞系并不是严格的贴壁依赖细胞(anchoragedependentcell,ADC),因此既可以进行单层细胞培养,又可以进行悬浮培养。杂交瘤细胞的单层细胞培养法是各个实验室最常用的手段,即将杂交瘤细胞加入培养瓶中,以含10-15%小牛血清的培养基培养,细胞浓度以1×106-2×106/ml为佳,然后收集培养上清,其中单抗含量约10-50ug/ml。显然,这种方法制备的单抗量极为有限,无疑是不适用于单抗的大规模生产。要想在体外大量制备单抗,就必须进行杂交瘤细胞的大量(高密度)培养。单位体积内细胞数量越多,细胞存活时间越长,单抗的浓度就越高,产量就越大。
不过也有用 HRP标记的protein a或g的,前提是你跑的样里没有IgG之类的东西。
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