The largest database of trusted experimental protocols

2 protocols using rabbit anti setdb1

1

Immunocytochemistry of Cell Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
After 3 h in culture on the microprinted PDMS layers, cells were fixed with 4% PFA at room temperature (RT) for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min at RT and washed 3 times in PBS. The immunochemistry process was performed following these steps: the fixed cells were incubated for 30 min in a blocking solution (0.2% tween, 1% Bovine Serum Albumine BSA, 1% Foetal Bovine Serum FBS) at RT, then the primary antibody was incubated for 40 min, washed three times in PBS-tween 0.2%, the secondary was incubated for 30 min, washed three times with PBS. The nucleus was then labeled with 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at RT and the actin filaments (F-actin) with SiR-actin (Spirochrome) at 100 nM in PBS overnight at 4 °C. The antibodies used for the immunochemistry were: mouse anti-Flag (Sigma Aldrich M2 F3165) 1/1000, rabbit anti-SMYD3 (Abcam ab187149) 1/300, rabbit anti-TAZ (Cell Signaling Technology 4883) 1/500, mouse anti-YAP (Santa Cruz Biotechnology 101199) 1/250, rabbit anti-Pan tri-methyl lysine (Cell Signaling Technology #14680) 1/1000, rabbit anti-Pan di-methyl lysine (Cell Signaling Technology #14117) 1/1000, DNase 1 Alexa Fluor 594 (Life Technologies) 1/1000, Rabbit anti-SETDB1 (Santa Cruz Biotechnology sc-66884) 1/200.
+ Open protocol
+ Expand
2

Testicular Cell Lysis and Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Testicular cells were lysed with RIPA (Beyotime Institute of Biotechnology, Jiangsu, China) that contained 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1% triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA and protease inhibitors, and were centrifuged at 12,000 g for 10 min at 4°C. Cell lysates were mixed with SDS loading buffer and boiled at 100°C for 5 min. Cell lysates were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). The membranes were incubated with the following primary antibodies: rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-SETDB1 (1:500; Santa Cruz Biotechnology; sc-66884), rabbit anti-EZH2 (1:1000; Cell Signaling Technology; 4905), mouse anti-beta-ACTIN (1:2000; CWBIO; CW0096M) and rabbit anti-H3 histone (1:2000; Millipore; 06-755). Secondary antibodies were horseradish peroxidase-linked anti-rabbit or anti-mouse antibody (1:5000; Abcam). The membranes were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Bio-Rad).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!