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frequently asked questions
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How to pretreat antibody samples from serum, ascites, culture supernatant and other sources?
The most common pretreatment method is precipitation method. Lipoprotein in ascites is usually precipitated with dextran sulfate, or when pH=7, b-lipoprotein and globulin are precipitated with PVP. If the ascites sample is too viscous during sample loading, the sample can be diluted with sample loading buffer. For albumin and other miscellaneous proteins, the fractional precipitation method can be used to remove them, such as ammonium sulfate precipitation, caprylic acid precipitation, etc. The salt ions can be removed and the buffer solution can be replaced by desalination column or dialysis.
How to select filler for purification of IgM and IgY that do not combine with Protein A or G?
(1) Usually, precipitation method combined with gel filtration method is used to purify IgM, but for a large number of purified antibodies, this scheme is cumbersome and low yield.
(2) Protein L affinity filler is used for specific binding κ Light chain, which is very useful for purifying antibody fragments and complete antibody IgG, IgM and IgA.
(3) The thiophilic filler is a kind of universal antibody purification method, which is based on the specific interaction between its ligand and the disulfide bond between the antibody. It can be adsorbed and eluted under neutral conditions and has high protein activity after purification.
How to purify antibody Fab fragment?
(1) Protein L can specifically bind antibody κ Light chain, so as long as the Fab clip contains κ Light chain, protein L filler can be selected for purification.
(2) Protein G can specifically bind Fc fragments of IgG, and can also specifically bind some antibody Fab fragments with low affinity.
(3) Protein A only binds Fc fragments of IgG. In the flow through mode, pure IgG was enzymatically hydrolyzed and passed through Protein A filler, Fab was in the flow through peak, and Fc fragment was bound to the filler.
How to improve the antibody recovery rate?
(1) Replace the filler with higher affinity.
(2) For Protein A filler, the combined pH can rise to 8-9.
(3) After the filler is used for many times, the residue increases and the loading decreases. It is recommended to clean the filler with 6M guanidine hydrochloride or 70% ethanol. For alkali resistant filler, 0.1-0.5M NaOH can be used for cleaning.
Why is there no correct band in the eluent?
(1) First, check whether the sample is bound, whether the antibody species and subtypes have affinity with the affinity filler, whether the pH of the binding buffer is neutral, and whether the antibody is reduced during the flow through. The original sample and the flow through can be analyzed by SDS-PAGE electrophoresis at the same time.
(2) Then check whether the sample is firmly bound to the filler and does not elute. It is recommended to use a buffer with a lower pH for elution.
(3) If the antibody is hydrolyzed under acidic conditions after elution, neutralizer or arginine and other protective agents can be added into the collection pipe in advance, or fillers that can be eluted under near neutral conditions can be selected.
What if the purity of antibody after elution is not high?
(1) Is the cleaning complete? The final washing solution can be analyzed by SDS-PAGE electrophoresis.
(2) Properly increase the salt ion concentration when binding, or add low concentration non-ionic detergent in the washing solution to reduce the non-specific adsorption.
(3) Sample pretreatment before loading to remove some impurities.
(4) Two steps of purification are adopted, first affinity chromatography is used for purification, and then ion exchange chromatography is used to remove HCP, fallen Protein A, and some polymers to obtain antibodies with higher purity.
How to avoid the interference of IgG in culture medium such as calf serum when IgG is expressed in vitro?
(1) Serum can be pretreated with Protein G, and IgG can be removed before culture. Or select the serum with low IgG content.
(2) The sample was first purified with Protein A or G, and the antibody obtained was then removed by hydrophobic chromatography.