Primary Antibodies Frequently Asked Questions topic (II)

Q1:What are the differences between WB, IHC, IP/ChIP type antibodies?

Answer:

WB: The most commonly made type at present, the proteins recognized by WB are the proteins with linear structure after heating denaturation.

IHC: A fixation step is required in IHC, in order to maintain the morphology and structure of the cells as consistent as possible. The fixation of this chemical causes the protein to denature and solidify, which is different from the natural state of the protein, but it is different from the linear structure of the WB.

IP/ChIP: This type of experiment uses antibodies to bind proteins in their physiological state, so it is best to use antibodies made from purified natural proteins for IP and ChIP.

Q2:Do recombinant antibodies converted from monoclonal perform identically to the original monoclonal?

Answer:

There may be a slight variation due to the production of the monoclonal antibody in a different system, but the two antibodies generally have similar applications and performance specifications.

Q3:What grade BSA is recommend for use in ELISA and ELISpot Development Systems?

Answer:

The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended.

Q4:What is a chimeric antibody?

Answer:

Chimeric antibodies are antibodies that made up of domains from different species. For example, the Fc region or all the constant region domains of a mouse monoclonal antibody may be replaced with constant region domains of a human antibody.

Q5:Why is the Western blot band size observed with a cell or tissue lysate sample different from the reported predicted molecular weight?

Answer:

 The "predicted" molecular weight is based entirely on amino acid sequence (or the size of the protein). However, there are other factors which may play a part in determining the observed size of the protein in a Western blot.Protein modification:  Post-translational cleavage where a larger pro-form of the protein is cleaved into a smaller active form which decreases its size in the gel or post-translational modification where a protein becomes glycosylated (N or O linked sites), phosphorylated or ubiquitinated increasing its size in the gel.The overall net protein charge determined by the amino acid composition may affect the migration speed through the negatively charged SDS of the gel.Trimerization, or dimerization creating multimeric proteins.  The use of  reducing conditions will help to eliminate these interactions.Alternative splicing may produce differently sized proteins from the same gene. 

Q6:Why might a recombinant protein have a different observed molecular weight in a Western blot compared to a cell or tissue lysate sample?

Answer:

There are a number of reasons why differences may be observed.The recombinant protein may only contain part of the mature amino acid sequences. Post translational modifications may be different in the recombinant protein than in the natural protein. For example, recombinant proteins produced in E. coli will not be glycosylated, which would make them appear smaller on a Western blot than the natural protein if there are glycosylation sites available in the sequence.The recombinant protein may contain a tag (e.g. His, Fc, or a basic or acidic tail). Check the Source section of the recombinant protein datasheet to check for additional tags or sequences that may be present.

Q7:How should I load antibodies?

Answer:

Preferably, it can be divided according to the usual dosage of an experiment. If the experiment can be completed within a week, the required amount of antibody can be divided and stored at 4℃. If the experiment period is long, 50% glycerol can be added and stored at -20℃. The remaining amount can be divided and stored at -80℃, which can be stored for 2-3 years.

Q8:How should I aliquot my antibody?

Answer:

The size of the aliquots will depend on how much one typically uses in an experiment. The aliquots should be no smaller than 10µl. The smaller the aliquot is, the more the concentration is affected by evaporation and adsorption of the antibody onto the surface of the vial.

Q9:Does this Biosimilar antibody have the same sequence as the therapeutic antibody?

Answer:

The Biosimilar antibody has the same variable region and constant region sequence as the therapeutic antibody.

Q10:Why is Western blot listed as an application on a hybridoma version of a monoclonal antibody, but not for the corresponding recombinant conversion of that antibody?

Answer:

We choose not to add a Western blot claim on the recombinant conversion of a monoclonal antibody if the only sample tested was a recombinant protein and not a lysate.