Is there a simple way to perform label-free quantification of Host Cell Proteins?
Question: I find it difficult to get an accurate label-free quantification of Host Cell Proteins with LC-MS. It also seems that...
Quickly compare HCP in different process steps, without setting up an ELISA
Question: I have spent days trying to set up a useful ELISA assay for detecting Host Cell Proteins (HCPs) in my process samples. I'm...
How to identify peaks observed by UV-HPLC in stability studies
Question: We have developed a Reversed-Phase (RP) UV- HPLC chromatography method. It is used for stability studies of our new pharmaceutical protein. How do we...
What is SWATH and why is it an advantage for Host Cell Protein analysis?
Host Cell Proteins (HCPs) in protein biopharmaceuticals can be analyzed by mass spectrometry - for both identification and quantification of...
4 reasons why Amino-terminal sequencing of mAbs should be done by Edman degradation
Question: How do I obtain the N-terminal sequences of the heavy chain and light chain of my monoclonal antibody? Answer: mAb N-terminal sequence...
GPMAW lite – Protein Molecular Weight Calculator (and much more)
Simple bioinformatics calculations on specific protein sequences When working with proteins you often need to make simple bioinformatics calculations on a specific...
How to: Make sure that Ile/Leu are correctly assigned by de-novo sequencing of your antibody
Question: I was told that de-novo sequencing by mass spectrometry is not able to distinguish between Ile and Leu residues in my...
What purity is required for de-novo sequencing of my antibody?
Question: My monoclonal antibody is not 100% pure, what is the purity required for de-novo sequencing? Answer: We typically observe two types of protein impurities in...
Antibody de-novo Sequencing by Mass Spectrometry: How to get 100% sequence coverage
Question: Is it possible to perform a de-novo sequencing of my purified monoclonal antibody without the hybridoma cells? Answer: Yes! – Your purified antibody can...
Reasons why you need accurate concentration determination of your protein standards
How do I quantify my protein standard? Have you ever questioned the quantitative data you get from Bradford, Lowry or BCA protein quantification...
How to determine the Molar Extinction Coefficient of your protein (in 3 small steps)
Question: I have a purified protein that I would like to quantify accurately in my lab using 280 nm UV measurement...
Identification of small host cell proteins (low Mw HCPs)
Question: Could you suggest an analysis method to detect low Mw HCPs (small HCPs) below 20 kDa? I work on a drug...
4 things to remember about Amino Acid Analysis of proteins and peptides
Amino acid analysis is a method to determine the absolute amounts of individual amino acids in a sample. The method...
1D SDS PAGE versus 2D PAGE. I’m considering 2D electrophoresis, any recommendations?
Question: I’m interested in isolating and identifying cell surface receptor proteins, typically with high Mw, glycosylated and with transmembrane domains. I’m...
3 steps to identify and quantify HCP in a drug substance homolog from a host cell
Question: My HCP antibodies show a reaction towards a drug substance band in my Western blot. Is it possible that it is...
How should I prepare my samples for 2D electrophoresis, and how much protein should I load to get protein identification by mass spec?
How should I prepare my samples for 2D electrophoresis, and how much protein should I load to get protein ID...
How to increase protein concentration before loading onto 1D SDS PAGE
Question: I have another question: Is it ok if I precipitate my samples with TCA prior loading to the SDS-Gel? If not, how...
Common protein contaminants observed in mass spectrometric protein ID
Questions: Is the keratin identified by the protein ID service in my 1D gel band a contamination? Or is it...
Effective 1D/2D PVDF staining protocols (for N-terminal sequencing)
For N-terminal Edman sequencing SDS PAGE separated proteins can be electroblotted onto PVDF membranes. After PVDF staining You may cut...
Mass spectrometric identification of proteins detected in Western blots
Question: Dear Sir, We have an unknown protein recognized in Western blot by autoantibodies that we need to identify. Is it...
What buffer to use for MALDI mass spectrometry analysis of a protein?
Question: What buffer should I use for MALDI Mass Spectrometry analysis of my protein sample? Answer: In general, MALDI-MS is more tolerant...
Useful Protein Molecular Weight Calculator kDa
Protein molecular weight calculator kDa The rule of thumb for conversion of microgram amounts into picomole amounts at different protein molecular...
What does the Mascot Score mean?
Question: I just got my protein identification report. What does the Mascot Score mean? Answer: The Protein Identification report contains a list of proteins that have...
Why is my protein’s N-terminal blocked (and what can I do about it)?
N-terminal sequencing is a requirement according to the ICH Q6B Guideline. This includes structural characterization of recombinant proteins for clinical...
Identification of proteolytic degradation of recombinant protein
Question: I purified a recombinant protein and analyzed it by 1D SDS PAGE. In addition to the intact protein (52...
Guide to identification of novel proteins not present in public databases
Question: I recently used Alphalyse's protein identification service to analyze my protein. The sample gave me very good MS data. However it...
Preparing samples for N- and C-terminal sequencing by MALDI ISD?
Question: I am interested in your N and C-terminal sequencing using MALDI ISD. How do I prepare the sample for...
Assessing the stability of a protein drug substance and formulated drug product
After protein purification, an important issue is the stability of a protein drug substance and formulated drug product. Stability assays...
How to optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing
How do I optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing? Our 12 kDa protein separates well on...
3 Techniques for N- and C-terminal sequencing of proteins
Where does the amino acid sequence start and where does it end? The terminal amino acid sequences are important for the...
N- and C-terminal protein sequencing using MALDI ISD
Structural characterization of purified natural and recombinant proteins traditionally includes N-terminal Edman sequencing to confirm 5-15 amino acid residues from...
Which databases and organisms are used for mass spec protein ID service?
Question: I would like to identify proteins from an organism where the genome has just been sequenced. How many proteins are...
Mass Spectrometry for Host Cell Protein monitoring and control in pharmaceutical process development
Mass spectrometry based Host Cell Protein identification and quantification Here the Mass Spec Host Cell Protein (HCP) assay is applied for...
Protocol for silver staining of gels optimized for mass spectrometry and protein identification
Problems arising from using commercial staining kits: Scientists use silver staining for sensitive detection of proteins separated by 1D and 2D...