FAQs
The most commonly used pretreatment method is precipitation. For lipoproteins in ascites, dextran sulfate is often used, or PVP can be used to precipitate β-lipoprotein and globulin at pH 7. If the ascites sample is too viscous during loading, dilute it with the loading buffer. To remove impurities like albumin, you can use graded precipitation methods such as ammonium sulfate or caprylic acid precipitation. Desalting columns or dialysis can be used to remove salt ions and replace buffers.
(1) Protein L specifically binds to the κ light chain of the antibody. Therefore, if the Fab fragment contains a κ light chain, Protein L resin can be used for purification.
(2) In addition to binding to the Fc fragment of IgG, Protein G can also bind with low affinity to certain antibody Fab fragments.
(3) Protein A binds exclusively to the Fc fragment of IgG. In flow-through mode, pure IgG is enzymatically hydrolyzed and passed through Protein A resin. The Fab fragment is collected in the flow-through peak, while the Fc fragment binds to the resin.
(1) IgM is typically purified using precipitation combined with gel filtration. However, this method is cumbersome and yields low results for large-scale antibody purification.
(2) Protein L specifically binds to κ light chains, making it useful for purifying antibody fragments and complete antibodies such as IgG, IgM, and IgA.
(3) Hiophilic-superflow resins utilize the specific interaction between their ligands and disulfide bonds in antibodies, adsorption and elution occur under neutral conditions, resulting in purified proteins with high activity. This method is a common option for antibody purification.
(1) Replace a higher affinity resin.
(2) For Protein A resins, increase the binding pH to 8-9.
(3) Residue build-up and decreased loading capacity occur with resins repeated use. Clean the resin with 6M guanidine hydrochloride or 70% ethanol. For alkali-resistant resins, use 0.1-0.5M NaOH for cleaning.
(1) Check Washing Completeness: Perform SDS-PAGE electrophoresis analysis on the final washing solution to ensure complete washing.
(2) Increase the salt ion concentration during binding, or add a low concentration of non-ionic detergent to the washing solution to reduce non-specific adsorption.
(3) Pre-treat the sample before loading to remove some impurities.
(4) Use Two-Step Purification: First, use affinity chromatography, followed by ion exchange chromatography to remove HCP, detached Protein A, and some aggregates, to obtain higher purity antibodies.
(1) First, check whether the sample is bound, whether the antibody species and subtype have affinity with the affinity filler, whether the pH of the binding buffer is neutral, and whether the antibody in the flowthrough is reduced. The original sample and the flowthrough can be run on SDS-PAGE electrophoresis for analysis.
(2) Check whether the sample is too firmly bound to the filler and cannot be eluted. It is recommended to elute with a buffer with a lower pH.
(3) Check whether the antibody is hydrolyzed under acidic conditions after elution. Neutralizing solution or protective agents such as arginine can be added to the collection tube in advance, or fillers that can be eluted under near-neutral conditions can be selected.
(1) The serum can be pretreated with Protein G to remove IgG before culture. Alternatively, serum with a lower IgG content can be selected.
(2) The sample is first purified with Protein A or G, and the resulting antibody is then subjected to hydrophobic chromatography to remove bovine IgG.
8. How should cell lines be packaged for shipment?
Ship cryopreserved vials via dry ice.
9. Do you require mycoplasma testing before sending cell lines?
Yes, all cell lines must be tested negative for mycoplasma within 30 days prior to shipment.
10. How do you handle cell lines with antibiotic resistance?
Clearly state the type and concentration of antibiotics.
11. How long does it take to supply cell lines after receiving a request?
Cryopreserved vials ship takes a few weeks, while actively growing cultures may require 1–2 weeks to reach proper confluence and quality check standards.