4. 加入primary antibody在cold room 4度过夜;(一般是1:1000稀释,如果非特异性条带过多就1:2000,如JARID2) 5. 用TBST洗3次,每次5分钟; 6. 加入secondary antibody在室温结合1小时;(一般是1:5000稀释,如果信号太弱就1:4000) 7. TBST洗4次,每次5分钟; 8. Image blot成像,Odyssey; 2023年06月23日 蛋...
Figure 2 – Effect of Varying Primary Antibody Concentration, % Milk in Antibody Solution, and NaCl Concentration in Blotting Buffer: Western blot optimization using R&D Systems rabbit anti-human PTP1B affinity-purified polyclonal antibody (Catalog #AF1366). Human HeLa (Lane 1), MCF-7 (Lane 2)...
be problematic for immunoprecipitation (IP) and pull-down assays prior to western blot. When it’s crucial to preserve protein-protein interactions or to minimize denaturation, you should use a buffer without ionic detergents (eg SDS) and ideally without non-ionic detergents (eg Triton X-100). ...
同时，选择适当的抗体稀释度和优化实验条件也是关键。Figure 6: Serial Dilution of Secondary Antibody For optimal results, it is recommended to dilute the antibody according to the suggested concentration. Shorter incubation times for the secondary antibody can be employed, with a suggested incubation perio...
Primary antibody concentration.0.1-0.5 μg/mL. Adjust antibody concentration from 0.05 up to 2.0 μg/mL to obtain desired signal strength and low background. Sample concentration.10-20 μL of cell lysates at 1x107cells per mL. (This is typically equivalent to 15-30 μg of total protein)...
1、细胞与分子生物学实验教学平台细胞与分子生物学实验教学平台 Western BlotWestern blot (immunoblotting)1. What is Western blot? A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. So called since it has some similarity to a Southern blot. 2. Why ...
Optimizing your antibody concentration is even more important when using high-sensitivity ECL substrates. A common misconception is that increasing the concentration of the primary and secondary antibodies will lead to a better signal. However, this is not always the case, especially when using high-...
3. Block the blot with 10% nonfat dried milk (NFDM) freshly made in TTBS; rock on a rotating shaker for 15 min. at room temperature or overnight at 4°C. 4. Rinse the blot 3 times in TTBS. 5. Probe with primary antibody in TTBS/1% NFDM for 1 hr. at room temperature. Primary...
If differentiation of the primary antibody host species is difficult, consider antibodies that are of a single specific antibody class (IgM, IgG, etc.) or isotype (IgG1, IgG2a, etc.), and a relevant secondary antibody that recognizes...
The primary antibody immunoprobing solution was prepared by diluting stock solutions of primary antibodies in 2% (w/v) BSA/TBST solution to achieve an antibody concentration of 0.05 µg/µL (single antibody). Primary antibodies used were EpCAM (Abcam, AB32392), VIM (Abcam, AB8978, ...