Western blot was performed as previously described [42 (link)]. Briefly, cells were lysed in the radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) Sodium deoxycholate, 0.1% (w/v) SDS] with protease inhibitors cocktail (Sigma) added freshly. The lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore), which were blocked in 5% milk for 1 hour and then probed with antibody against TCTN1(1:200; ab105381; Abcam), or actin (1:4000; M20010; Abmart) as a loading control. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized on G: Box Chemi XR5 (Syngene).
M20010
Manufactured by Abmart
Sourced in United States
The M20010 is a laboratory equipment product designed for general scientific applications. It serves as a core functional device to support various laboratory operations. The product's specifications and capabilities are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Ab105381, by Abcam (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
G box chemi xr5, by Syngene (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Enhanced chemiluminescence plus kit, by Merck Group (1 mentions)
Kgp602, by Keygen Biotech (1 mentions)
Kgp603, by Keygen Biotech (1 mentions)
Kgaa3d5, by Keygen Biotech (1 mentions)
Kgp250, by Keygen Biotech (1 mentions)
Ab52930, by Abcam (1 mentions)
Imager 600, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
3 protocols using m20010
1
Western Blot Protein Detection Protocol
2
Whole-Cell Lysis and Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from tissues was lysed in a whole‐cell lysis assay (Cat. KGP250, KeyGen BioTECH, China) containing protease inhibitors (Cat. KGP603, KeyGen BioTECH, China) and phosphatase inhibitors (Cat. KGP602, KeyGen BioTECH, China). Then, 10 µg of protein per sample was resolved by 10% SDS‐PAGE and transferred onto PVDF membranes. The membranes were first incubated overnight at 4°C in BSA in TBS containing 0.05% Tween 20 with primary antibodies against β‐actin (1:5,000, Cat. M20010, Abmart, USA) and PMS2 (1:1,000, Cat. 66075‐1, Proteintech, USA), followed by incubation with secondary antibodies (Cat. KGAA3d5, KeyGen BioTECH, China) conjugated with horseradish peroxidase at room temperature for 1 hr. The protein bands were detected using an enhanced chemiluminescence plus kit (Millipore) according to the manufacturer's instructions.
3
Analyzing Protein Expression via Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples collected from mice or cell culture were lysed by RIPA buffer containing protease inhibitor cocktail (04693132001, Roche) on ice. Equal amounts of protein were electrophoresed in 12% SDS-PAGE gel and transferred to PVDF membranes. These membranes were incubated with primary antibodies of HIP2 (ab52930, Abcam) and actin (m20010, Abmart) at 4 ℃ overnight, followed by secondary antibodies incubation at room temperature for 2 h. Images were obtained by Amersham (Imager 600, GE) and quantified by Quantity One (version 4.6.2, Bio-Rad).
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
Revolutionizing how scientists
search and build protocols!