Protein preparation for mass spectroscopy was performed as previously described [19 (link)]. Protein samples were diluted in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer. RapiGest SF reagent (Waters Corp., Milford, MA) was added to the mix to a final concentration of 0.1% and samples were boiled for 5 min. Tris (2-carboxyethyl) phosphine (TCEP) was added to 1 mM (final concentration) and the samples were incubated at 37°C for 30 min. Subsequently, the samples were carboxymethylated with 0.5 mg/ml of iodoacetamide for 30 min at 37°C followed by neutralization with 2 mM TCEP (final concentration). Proteins samples prepared as above were digested with trypsin (trypsin:protein ratio - 1:50) overnight at 37°C. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37°C for 1 h followed by centrifugation at 13,000 g for 30 min at 4°C. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using Aspire RP30 desalting columns (Thermo Scientific, Waltham, MA).
Aspire rp30 desalting columns
Manufactured by Thermo Fisher Scientific
The Aspire RP30 desalting columns are designed for the rapid and efficient removal of salts, buffers, and other small molecules from protein samples. The columns use a size-exclusion resin to separate the protein of interest from contaminants, allowing for the recovery of purified protein samples.
Automatically generated - may contain errors
3 protocols using aspire rp30 desalting columns
1
Protein Preparation for Mass Spectrometry
2
Immunoprecipitation and Mass Spectrometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiment was performed as described previously24 (link),66 (link). In brief, 293T cells were transfected with Myc-tagged SRPK1 and wild-type Tip60 or HAT-deficient Tip60. The cells were then lysed, and immunoprecipitation was performed with the c-Myc antibody and Protein A/G agarose beads (Santa Cruz). The precipitated proteins were subjected to trypsin digestion (protein:trypsin in a weight ratio of 1:50) overnight at 37 °C. After extraction, the digested peptides were desalted by using Aspire RP30 desalting columns (Thermo Scientific) in vacuum. Subsequently, they were analysed by the high-pressure liquid chromatography tandem mass spectroscopy (LC-MS/MS). The collected data were analysed using MASCOT (Matrix Sciences) and Protein Pilot 4.0 (ABSCIEX) for peptide and modification identifications. Carbamidomethyl (C) was set as the fixed modification; acetylation (K), oxidation (M) and deamidation (N) were set as variable modifications.
3
Protein Digestion and Peptide Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were diluted in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer. RapiGest SF reagent (Waters Corp.) was added to the mix to a final concentration of 0.1% and samples were boiled for 5 min. TCEP (Tris (2-carboxyethyl) phosphine) was added to 1 mM (final concentration) and the samples were incubated at 37°C for 30 min. Subsequently, the samples were carboxymethylated with 0.5 mg/ml of iodoacetamide for 30 min at 37°C followed by neutralization with 2 mM TCEP (final concentration). Protein samples prepared as above were digested with trypsin (trypsin:protein ratio –1∶50) overnight at 37°C. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37°C for 1 h followed by centrifugation at 14000 rpm for 30 min at 4°C. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using Aspire RP30 desalting columns (Thermo Scientific).
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!