FAQ: Expression and Purification

Q: Why do I heat the lysed cells to 60C?

A: The rABs produced by the RAN are extremely temperature stable and heating to 60C assists in purification by precipitating E. coli proteins and degradation products prior to purification.

Q: What is the expected yield of the rAB’s?

A: We typically see expression levels between 1mg/L and 10mg/L.

Q: We can see bands around 25 kDa on the SDS-PAGE after rAB purification. What should we do?

A: These bands are often unpaired heavy or light chains. These may not necessarily reflect the quaternary rAb structure in solution, but may also represent excess heavy or light chain that did not pair properly. In some cases, these unpaired species will precipitate out of solution leaving the expected 50 kDa and this can be promoted by eluting in a smaller volume of elution buffer.

Alternately, in some cell lines, partial proteolysis or clipping may occur due to the presence of bacterial proteases during expression.  In this case, we recommend...

Q: Are your rABs biotinylated and why would you do that?

A: In some cases, more versatile detection schemes may be desirable. The rABs can be converted to include alternate tags and fusion proteins.

Q: Do all RAN rAbs have an Avi-tag?  

A: No. The presance of an Avi-tag can be determined by the vector type and is listed in the vector features. Vectors RH2.2-Avi, pSFV4, pSFV3 and pCW-FABAVI-LIC have the Avi-tag. Vectors pFab007, RH2.2 and pBL166 do not.

Q: How are rAbs that have the Avi tag expressed and biotinylated?

A: Site-specific biotinylation of rAbs is made facile by fusion to an Avi tag to the C-terminus of the rAB heavy chain and expression in a competent E. coli strain pre-transformed with a biotin ligase (BirA) expression construct and maintained by it’s chloramphenicol resistance. Media is supplemented with biotin and often expressed proteins are >90% biotinylated.

Q: I purchased an Avi-tagged clone but would like non-biotinylated antibody. Can’t I just express in a cell line that is not transformed with BirA?

A: While Avi-tagged rAb clones can be expressed in non-BirA transformed cell lines, endogenous biotin ligase will biotinylate the Avi-tag on the rAB and contribute to background signals in downstream assays using the biotin tag for detection. In this case the degree of biotinylation is variable and must be determined empirically.

Q: What is the maximal concentration of reducing agent I can use in my assay buffer?

A: We have tested DTT concentrations up to 100uM and would suggest titration to verify the maximal concentration for your specific rAB.

Q: I’ve tried using rABs for Western Blotting but can’t get a positive signal with a positive control.

A: Most RAN rABs are selected against folded antigen in the liquid phase. Often Western blotted proteins are either partially or fully denatured on a solid support and would not be expected to react with rABs to the folded protein. Nevertheless in some cases, antigens can at least partially refold and may react with antigen but this must be determined empirically.

Q: What ratio of rAB to target protein you use when setting us crystallization trials?

A: 1:1.2 usually works well and it is advised to run protein through gel filtration to isolate pure complex. Usually 30 min incubation time is sufficient for complex formation.

Q: What buffers can be used with rABs?

A: Most commonly used buffers are compatible with rABs.

Q: I can see one diffused band on the gel? Why is that?

A: Not enough protein. There are actually two bands running closely on the gel. Try increasing resolution of your separation and adjust amount you load.

Q: Why molar extinction coefficients for different rABs vary so much?

A: As for any protein, the extinction coefficient will depend in part on the specific composition of absorbing amino acid residues which can vary from antibody to antibody. For precise determination of rAb concentration, we recommend multiple assays (spectrophotometric, dye-binding and SDS-PAGE) and correlating the results for agreement.

Q: I usually use monoclonal antibody at 2000x dilution what dilution do you recommend?

A: We recommend users to titer the recombinant antibodies to determine the correct dilution in their application.

Q: Do you have antibody X available?

A: Please check our catalog. If we do not have it there you can contact us and we are open to discussing arrangements for antigen provision, selections and validation.

Q: The rAB is reacting with both it’s target protein and non-specific control proteins. Why?

A: Most likely, too high a rAB concentration is being used. Optimal concentrations for rABs have to be determined for a given application. For custom applications, we recommend tirating in rAB on both target and non-specific control protein (where possible) to determine the optimal concentration.

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