Issues in Validation of Post-Translational Modifications.
Why are proteins, detected by mass spectrometry, not validated by site-specific antibodies?
The modified motif could be detected by mass spectrometry (MS) after affinity enrichment, then the modified protein should be affinity-enriched for western-blotting validation, ELISA analysis with the the sequence-specific, modified peptide antibodies.
Can the sequece-specific modifed peptide antibody used for affinity-enrichment？
Only if this modified peptide antibody is able to immunoprecipitation (IP) the modified proteins.
Not all the peptide antibodies are able to perform IP studies, even if it could be utilized for Western blotting (WB).
By using the pan-specific modified amino acid antibodies to affinity-enrich all the modified proteins, you may selectively detect the target modified proteins by WB using modified sequence-specific antibodies.
For WB, you could only resolve maximum 100 ug/lane of crude proteins in SDS-PAGE, your target modified proteins in 100 ug of crude lysates may not be detectable and may also interfere with other abundant proteins, but you could enrich your target modified proteins only from 1-10 mg of crude proteins, the enriched modified proteins could readily be resolved in SDS-PAGE and become detectable level for WB.
Three trimethylated sites of histone H3 enriched with 100 uL of the pan trimethyl lysine specific antibodies (ICP0610) Agarose (1 mg of the antibodies) from 5 mg crude proteins, detected by WB with three site-specific antibodies.
PTM Proteomic Analysis Sample Preparation Scheme
Follow our schematic for consistent and accurateresults.
Biotin Antibody Agarose - ICP0615
Biotin is a vitamin, which binds strongly and irreversibly to avidin. Biotin is commonly used for labelling reagents. Anti-biotin antibody binds to biotin strongly but reversibly, the binding could be competitively eluted with free biotin.
10mg - $695
Biotin Antibody, HRP - ICP0612
This antibody has been used for direct competitive ELISA for quantization of d-biotin and western-blot analysis of biotinylated antibodies and proteins. The antibody reacts with free biotin and biotinylated antibodies and proteins.
1mg - $325
Biotin Antibody, FITC - ICP0613
This antibody was conjugated with FITC and has been used for indirect fluorescent staining studies. It reacts with free biotin and biotinylated proteins regardless of species.
1mg - $325
Biotinylated BSA - ICP0614
The biotinylated BSA is well recognized and binds strongly to the affinity purified anti-biotin antibodies.
5mg - $275
Acetyl Lysine Antibody, Agarose - ICP0388
Protein acetylation is a form of post-translational modification known to regulate many diverse biological processes. Affinity chromatography is one of the most efficient and rapid methods to enrich and purify the acetylated species for further MS/MS identification.
2mg - $550
5mg - $950
Methyl Lysine Antibody, Agarose - ICP0588
Protein methylation is a form of post-translational modification known to regulate many diverse biological processes. Affinity chromatography is one of the most efficient methods to enrich and purify the methylated species for further MS/MS identification.
5mg - $895
Trimethyl Lysine Antibody, Agarose - ICP0606
Rapid isolation and purification of the trimethylated species for proteomic profiling of the trimethylated proteins in different stages of development, disease, and signal stimulation, and identification of the protein trimethylation site.
5mg - $650
Phospho-Tyrosine Antibody, Agarose - ICP9804
This tool is essential for proteomic profiling of the phosphorylated proteins in different stages of development, disease, and signal transduction, as well as identifying the precise site of phosphorylation in a phospho-protein.
5mg - $650
Protein A Agarose - ICP1001
The immobilized protein A on the beaded agarose could be utilized for the immunoprecipitation and purification of both polyclonal and monoclonal antibodies from mammalian species. Protein A is very heat and acid stable.
2mg - $150