Mycoplasma are small bacteria that are one of the most prevalent microbial contaminants of mammalian cell cultures. Mycoplasma contamination can alter the growth and metabolic characteristics of cultured cells and lead to unreliable experimental results. Mycoplasma contamination often goes unnoticed as commonly no visible changes to medium (pH or turbidity) or cellular morphology occur.
Mycoplasma are small bacteria that are one of the most prevalent microbial contaminants of mammalian cell cultures. Mycoplasma contamination can alter the growth and metabolic characteristics of cultured cells and lead to unreliable experimental results. Mycoplasma contamination often goes unnoticed as commonly no visible changes to medium (pH or turbidity) or cellular morphology occur.
The CycleavePCR Mycoplasma Detection Kit enables the specific detection of Mycoplasma species from cell-culture samples using real-time PCR amplification of the Mycoplasma 16S rRNA gene. Detection of at least ten species in the genus Mycoplasma, including species commonly found in cell-culture media (M. arginini, M. hominis, M. hyorhinis, M. orale, M. salivarium, M. fermentas, M. bovis, M. arthritidis, M. pirum, and M. pneumoniae) and one species in the genus Acholeplasma (A. laidlawii) has been confirmed.
This kit enables amplification products to be detected by cycling-probe technology, a highly sensitive detection method that uses an RNA/DNA chimeric probe and RNase H. One end of the probe is labeled with a fluorescent moiety and the other end with a quencher. When intact, this probe does not emit fluorescence due to the action of the quencher. However, when it forms a hybrid with the complementary sequence of an amplification product, RNase H cleaves RNA in the chimeric probe, resulting in strong fluorescent signal emission. The amount of amplified product can be monitored by measuring the intensity of emitted fluorescence.
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我觉得封闭这一步时间长短多少并不能减少背景…背景过深多半还是抗体浓度过大…不管一抗还是二抗过大都会使背景加深…一抗的浓度不一定是条带清晰就是最佳了…联系进一步优化一下
这个主要还是抗体的问题,WB最主要的还是要选择一个特异性较好的抗体,抗体是整个实验的重中之重
内参抗体:
Beta-Actin mAb (1C7) 细胞总蛋白 42 kD Abbkine A01010 小鼠源单克隆
Beta-Actin mAb (1C7) , HRP 细胞总蛋白 42 kD Abbkine A01015 小鼠源单克隆,HRP偶联
GAPDH mAb (2B5) 细胞总蛋白 36 kD Abbkine A01020 小鼠源单克隆
GAPDH mAb (2B5) , HRP 细胞总蛋白 36 kD Abbkine A01025 小鼠源单克隆,HRP偶联
Beta-Tubuline mAb (3G6) 细胞总蛋白 55 kD Abbkine A01030 小鼠源单克隆
Plant Actin mAb (3T3) 植物细胞总蛋白 42 kD Abbkine A01050 小鼠源单克隆
PCNA mAb (1D7) 细胞核蛋白 28 kD Abbkine A01040 小鼠源单克隆
COX IV mAb (14Y2) 细胞线粒体总蛋白 16 kD Abbkine A01060 小鼠源单克隆
Histone H3 mAb (2D10) 细胞核蛋白 18 kD Abbkine A01070 小鼠源单克隆
标签抗体(Tag Antibody)可用于检测各种商品化表达载体上的标签序列(如:Myc、Flag、His、GST、HA等),籍以分析目的蛋白的表达含量及其功能;
标签抗体:
Anti-Biotin Antibodies
Anti-Dye Antibodies
Anti-FITC Antibodies
Anti-Fluorescent Protein Antibodies
Anti-HRP Antibodies
Beta Galatosidase Antibodies
FLAG Tag Antibodies
GST Tag Antibodies
HA Tag Antibodies
His Tag Antibodies
Myc Tag Antibodies
TAP Tag Antibodies
V5 Tag Antibodies
β-Actin作为内参是得到了公认的,这是针对大多数组织和细胞来说的,它广泛分布于细胞浆内,表达量非常丰富。Beta-actin由375个氨基酸组成,分子量大小为42-43kDa左右。
β-actin的蛋白水平通常不会发生改变,因此被广泛用于Western时上样量是否一致的参照,也常被用于免疫染色观察细胞的微丝结构。在用作Western的参照时,Actin抗体和Tubulin抗体的主要不同之处在于两者所识别蛋白的分子量不同,这样可以选择合适的参照在同一块胶同一张膜上实现同时检测目标蛋白和参照蛋白。向左转|向右转
因为这个实验用到了抗体,抗体是特异识别某一种蛋白的,所以只要能检测到信号,说明样品中有该蛋白存在,如果有相应的对照,也能半定量说明该蛋白的量如何。
虽然没有具体用过检测这个抗原的抗体,但是如果希望抗体特异性好点,能检测丰度低的蛋白,还是选Abcam吧,Santa有时候全凭人品了,内参抗体什么的还能凑活,其他的比较勉强。
GAPDH
万能内参
主要看你的目标蛋白的分子量多大,“目标蛋白要与内参蛋白的分子量差异大!”
方便检测
核蛋白,有很多种,分子量不同
膜蛋白,有很多很多种,分子量不同
浆蛋白,有成千上万种,分子量不同。
具体实验,具体目的蛋白,具体的内参。
一般可与大鼠、小鼠、人、兔均有交叉反应,即可作为上述任一来源蛋白的内参。
Actin分子量为42kD,为肌动蛋白
GAPDH分子量为36kD,为磷酸脱氢酶