How does the kit work?
The columns in the Pierce Phosphoprotein Enrichment Kit contain a proprietary metal that interacts with negative charges from phosphate groups. The optimized buffer conditions enable specific capture of phosphoproteins from complex biological samples.
Will the columns bind phosphoproteins modified on tyrosine residues?
Yes. The Phosphoprotein Enrichment Columns bind phosphotyrosine, phosphoserine and phosphothreonine residues.
How many samples can I process and what is included in the kit?
Each of the 10 columns in the kit can process a single sample containing up to 4 mg of total protein. The kit includes Lysis/Binding/Wash Buffer, CHAPS, Elution Buffer and 10 Pierce Protein Concentrators (7 ml/9K) for concentrating the enriched samples.
What type of yield should I expect?
Typical yields are 10-25% of the total protein, depending on cell stimuli, cell type and binding characteristics of the specific phosphoproteins.
What other type of lysis buffer can I use?
You can use RIPA buffer to lyse cells; however, dilute the lysate with the Lysis/Binding/Wash Buffer included in the kit for compatibility with the column.
Do I need phosphatase inhibitors?
Phosphatase and protease inhibitors are not required. If desired, add inhibitors to the Lysis/Binding/Wash Buffer as indicated in the product instructions.
Are samples containing EDTA or reducing agents compatible with the kit?
No. Chelating agents such as EDTA and EGTA will cause metal to leach from the column and hence, reduce binding. Reducing agents such as DTT, 2-mercaptoethanol and TCEP also reduce binding to the column. Remove incompatible substances from the sample by desalting before applying to the column.
Can I reuse or recharge the columns?
No. For best results, use the column only one time.
Can I recharge the column?
No. The metal and procedure used to charge the columns are proprietary.