FLS were lysed in 1 × NEBuffer (New England Biolabs) supplemented with 1 mM MnCl2. Cell lysate was incubated on ice for 20 min, spun down, and supernatant was collected and incubated with 10,000 U/ml lambda (λ) phosphatase (New England Biolabs) at 30 °C for 30 min. Protein lysate was added to loading buffer and boiled at 95 °C for 5 min, and further processed for immunoblotting as described above.
Lambda λ phosphatase
Manufactured by New England Biolabs
Sourced in United States
Lambda (λ) phosphatase is a recombinant protein phosphatase that catalyzes the removal of phosphate groups from serine, threonine, and tyrosine residues in proteins. It is a highly active and versatile enzyme used in various applications to dephosphorylate proteins.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using lambda λ phosphatase
1
Phosphatase-Mediated Protein Dephosphorylation
2
In vitro Dephosphorylation Assay of KAP1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro dephosphorylation assay was performed as described (Lee et al., 2010 (link); 2014 (link)). PP4IP-expressed or -knockout HCT116 cells were transfected with Flag-empty or Flag-PP4C plasmids and collected after 48 h. 293T cells were transfected with FH-KAP1 for 48 h, incubated with 10 μM CPT for 1 h and followed by further incubation for 1 h after release. HCT116 and 293T cells were immunoprecipitated using Flag antibody-agarose beads. Immunoprecipitates were incubated with buffer containing 50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35 for 1 h at 30°C in ThermoMixer C (Eppendorf, Germany). Lambda (λ) phosphatase (New England Biolabs, USA) serves as a control. After incubation, samples were resolved on SDS-PAGE, and the relative phosphatase activity was determined using antibody against anti-KAP1 pSer824 as the loss of FH-KAP1 phosphorylation immunoreactivity.
3
Phosphatase Treatment of Immunoprecipitated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation was performed as described above with the following changes: The beads were washed with 1× TBS three times and proteins were eluted with 55 μl of 100 μg/ml 3xFLAG peptide (Sigma) in 1× TBS. 20 μl of eluate was combined with 2.5 μl of 10 mM MnCl2, 2.5 μl of 10× PMP buffer (NEB), and 400 units of lambda (λ) phosphatase (NEB) for 24 hr at 4°C. The assay was halted by adding 5 μl of 6× Laemmli buffer with 10 mM DTT. Samples were boiled and 10 μl of sample was then used in SDS–PAGE.
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!