To detect phosphorylated proteins, 100 µg of total protein was loaded, and to detect immunoprecipitated proteins, the samples were loaded in equal volumes. Samples were separated in 4–8% SDS-polyacrylamide gel (SDS–PAGE) and then transferred electrophorectically to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked for 1 h at room temperature in Tris buffered saline (TBS, in mM: 137 NaCl, 20 Tris-HCl; pH 7.6) containing 0.1% Tween-20 (TBS-T) and 5% low-fat milk or 5% BSA to detect phosphorylated proteins. The membranes were incubated with the primary antibody (listed in Table 1 ) for 2 h at room temperature. After washing for 1 h in TBS-T, the membranes were incubated for 1 h at room temperature with the respective secondary antibody conjugated with alkaline phosphatase (GE Healthcare), prepared in TBS-T with 1% low-fat milk or 5% BSA. The immunoreactive bands were visualized with the Enhanced Chemi-Fluorescence system (ECF, GE Healthcare) and imaging system (Storm 860, Molecular Dynamics, GE Healthcare). Digital quantification of band intensity was performed using ImageQuant 5.0 software (Molecular Dynamics, GE Healthcare).
Alkaline phosphatase
Manufactured by GE Healthcare
Alkaline phosphatase is an enzyme found in various tissues, including the liver, bone, and intestines. It catalyzes the hydrolysis of phosphate esters under alkaline conditions. The quantitative determination of alkaline phosphatase levels can provide valuable information for the assessment of various medical conditions.
Automatically generated - may contain errors
2 protocols using alkaline phosphatase
1
Quantitative Western Blot Analysis
2
Genomic DNA Analysis of B. canis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA of B. canis was extracted from parasite-infected erythrocytes for Southern blotting. Five micrograms of DNA was used for each restriction digestion analysis using enzymes that did not cut the probe-specific sequence of BcMSA1 (BamHI, SalI) and BcSA1 (BamHI, SacI). Furthermore, the DNA was also digested with the restriction enzymes that cut a single site within the probe-specific sequence of BcMSA1 (PacI, KpnI and NaeI) and BcSA1 (NheI and KpnI), respectively. The digested DNA samples were submitted to 1 % (w/v) agarose gel electrophoresis. Then, the fractionated DNA was transferred to Hybond-N+ nylon membrane (Amersham-Buchler, Germany). The BcMSA1 and BcSA1 probes that were PCR-amplified using specific primers were directly labeled with alkaline phosphatase (GE Healthcare Bio-Science Co.) and hybridized following the manufacturer’s instructions. The filters were pre-hybridized at 56 °C for 6 h, and hybridization was carried out at 56 °C for 12 h in a hybridization oven with labeled probes of full-length of BcMSA1 and BcSA1, respectively. The blots were washed for several times. Chemiluminescence signals were generated using the CDP-Star detection reagent (GE Healthcare Bio-Science Co.).
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!