细胞上清超滤后用SBI试剂盒提外泌体中的问题

八戒在五湖

60mL用0.20滤膜过滤的细胞上清用100KD超滤管4000g，50min离心，收集大概600uL滤液
按5：1比例加入试剂，混匀，4℃过夜
1500g，30min离心，沉淀是深红色
PBS小心洗一遍，1500g，10min离心去上清，沉淀还是深红色，再用100uLPBS吹匀
跑western：CD9，CD63都没有条带
问题1：我在超滤过程中的时间是不是太长了，浓缩液体积太少，外泌体有没有可能挂在膜上？
问题2：沉淀是深红色正常吗，外泌体不应该是白黄色的么？

天道酬勤

1、外泌体会挂膜上的，有文献报道，要洗膜的
2、外泌体不应该是红色，红色是不是你培养基的颜色
3、western啥都没有，可能因为你PBS洗涤的问题，你洗涤过后exosome应该都在上清里了，你把它给弃去了。你洗涤后应该再次超速离心，那时的沉淀里才有exosome

八戒在五湖

天道酬勤 发表于 2017-9-6 15:19
1、外泌体会挂膜上的，有文献报道，要洗膜的
2、外泌体不应该是红色，红色是不是你培养基的颜色
3、western ...

超滤管的膜应该怎么洗，用枪吸PBS直接吹吗，我是想用试剂盒提，样品的体积要尽可能少一些，所以才要超滤浓缩

天道酬勤

Preparation of Urinary Exosomes by Nanomembrane Concentrator
Nanomembrane concentrators (Vivaspin 500, $3 for each 500 ul of tube; Vivaspin 4, $4 for
each 4 ml of tube or Vivaspin 20, $8 for 20 ml of each tube; Sartorius Inc., Goettingen,
Germany) were washed to remove glycerol and other preservatives by adding approximately
one volume of PBS buffer then centrifugation at 3,000 × g at room temperature (RT) before
processing urine samples. After centrifugation of urine samples at 17,000 × g at 4 ºC for 15
min, 0.5 ml, 1.5 ml or 10 ml of urine supernatant was added to a Vivaspin 500, 4, or 20,
respectively, nanomembrane concentrator and then centrifuged at 3,000 × g at 20°C for 10–
30 min. The urine proteins were recovered from the nanomembrane concentrator by two
methods: 1) A combined retentate (CR) was recovered by adding an equal volume of
unheated 2X solublizing buffer to the retentate while still in the concentrator and shaking at
RT for 30 min, or 2) sequentially recovering the proteins not bound to the nanomembrane
and adhering proteins: the retentate (R) was removed from the concentrator before adding an
equal volume of the pre-heated (95°C) 2X solublizing buffer (2X Laemmli buffer with 400
mM dithiotheritol (DTT)); the nanomembrane was subsequently washed to remove
remaining proteins that adhered to the nanomembrane by adding two volumes of pre-heated
1X solublizing buffer (HSW) into the concentrator and shaking the concentrator at RT for
10 min (Fig. 1). Urine exosomal proteins isolated by ultracentrifugation (200,000 × g
pellets) described previously(7) were used as positive controls. The combined retentate (CR)
method was used to isolate from the urine of 9 patients (male=5, female=4) with FSGS and
8 normal control volunteers (male=4, female=4). All of the urinary protein samples were
stored at −80°C until use.



https://www.ncbi.nlm.nih.gov/pmc ... /pdf/nihms42388.pdf

胡萝卜ss

你好，想请问一下你SBI试剂盒提取的外泌体，跑WB时，有没有出现同样的marker蛋白，外泌体的条带比细胞条带位置跑的低的情况？