Cells were synchronized at the G1/S transition by double thymidine block as above, but released into medium without CDK1 inhibitor. Cells were collected by trypsinization and lysed in RIPA cell lysis buffer [25 mM Tris-Cl pH 7.1,150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate, 0.1% SDS, Halt protease inhibitor (Pierce)]. Cells were incubated at 4 °C for 10 min and then centrifuged at 19000 × g for 15 min. The supernatant was removed, and the protein concentration was estimated using the DCA assay (Bio-Rad), and proteins were resolved by SDS-PAGE and immunoblotted with the appropriate antibodies.
Dca assay
Manufactured by Bio-Rad
The DCA assay is a laboratory equipment product designed for the quantitative determination of glycated hemoglobin (HbA1c) in human whole blood samples. It provides a reliable and accurate measurement of long-term blood glucose levels, which is a key indicator for the management of diabetes.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using dca assay
1
Cell Cycle Synchronization and Protein Analysis
2
Immunoprecipitation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in mammalian cell lysis buffer (50 mM Tris-Cl [pH 6.8], 150 mM NaCl, 0.5 % Nonidet P-40, Halt protease inhibitors [Pierce], and 1 mM DTT). To recover total protein (soluble and insoluble), sodium dodecyl sulfate (SDS) was added to a final concentration of 1% (Figure 1B, Supplementary Figure 2F and6A).
Cells were incubated at 4°C for 10 min and then centrifuged at 14,000 rpm for 15 min. The supernatant was removed, and the protein concentration was estimated using the DCA assay (Bio-Rad).
Myc or FLAG magnetic beads in a 1:1 slurry in mammalian cell lysis buffer was added to samples and rotated at 4°C overnight. A magnetic stand (Thermo Fisher) was used to wash the beads three times with lysis buffer. Beads were resuspended in 25 µl SDS sample buffer, boiled briefly, and processed for SDS-PAGE and immunoblotting. FLAG elutions were performed two times with the 250µg/ml 3XFLAG peptide (ApexBio) for 30 minutes at room temperature. Endogenous p97 was further immunoprecipitated (Bethyl) at 4°C overnight.
Beads were washed 3 times in lysis buffer and resuspended in Laemmli buffer and boiled briefly.
Cells were incubated at 4°C for 10 min and then centrifuged at 14,000 rpm for 15 min. The supernatant was removed, and the protein concentration was estimated using the DCA assay (Bio-Rad).
Myc or FLAG magnetic beads in a 1:1 slurry in mammalian cell lysis buffer was added to samples and rotated at 4°C overnight. A magnetic stand (Thermo Fisher) was used to wash the beads three times with lysis buffer. Beads were resuspended in 25 µl SDS sample buffer, boiled briefly, and processed for SDS-PAGE and immunoblotting. FLAG elutions were performed two times with the 250µg/ml 3XFLAG peptide (ApexBio) for 30 minutes at room temperature. Endogenous p97 was further immunoprecipitated (Bethyl) at 4°C overnight.
Beads were washed 3 times in lysis buffer and resuspended in Laemmli buffer and boiled briefly.
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!