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)...
2. Incubate the blot for 1 hour at 37℃, 2 hours at room temperature, or overnight at 4℃. 22)、一抗孵育 一抗用TBST1:1000稀释,将硝酸纤维素膜放入其中,37℃孵育1.5小时,(或4℃过夜)摇床摇动。 Probe with primary antibody in TTBS/1% NFDM for 1 hr. at room temperature. Primary antibody...
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 ...
Although not common in western blot, some applications require secondary antibodies that are adsorbed against other species to minimize recognition of endogenous immunoglobulins (such as probing mouse tissue lysate with an anti-rat primary antibody) ...
Primary antibody incubation: Dilute primary antibody to an appropriate concentration with blocking buffer. HRP-labeled secondary antibody: Soak the membrane in secondary antibody diluted with blocking buffer and shake for 2 hour at room temperature. Rinse the membrane breifly three time with an adequate...
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)...
5. Antibody staining 1. Block the membrane for 1-2 hours at room temperature using 5% blocking solution. 2. Incubate membrane with appropriate dilutions of primary antibody (Appendix 2) in antibody dilution buffer overnight at 4°C. 3. Wash the membrane in three washes of 1xTBST, 5 ...
Increase antibody concentration as necessary.Problem High background on the blot Possible Cause 1. Film overexposed or became wet during exposure 2. Short blocking time or washing intensity 3. High concentration of primary and/or secondary antibody 4. Protein is overloaded 5. Membrane, ...
Silver-stained 2-D gel of rat fibroblast cell line (left) and blot of the same gel (right), probed with a mouse monoclonal antibody and visualized using chemiluminescence on Immobilon® -P membrane. 15 of the membrane, and cing a weight Polyacrylamide Concentration on top of the filter ...