Analysis of Common
Experimental Issues
Focusing on common issues in core experimental technologies of life science research, we provide professional analysis and solutions for key pain points such as experimental procedures, result abnormalities, and operational difficulties, helping researchers efficiently troubleshoot and optimize experimental protocols.
Western Blot（WB）
Common issues include blurred bands, non-specific bands, absence of target bands, and band smearing, mostly associated with protein expression level, sample preparation, gel electrophoresis parameters, incubation conditions, and antibody conditions.
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Immunohistochemistry (IHC)
Problems such as weak/strong staining, high background staining, and inaccurate localization of positive signals are prone to occur, usually related to antigen retrieval methods, antibody dilution ratio, incubation time, and washing steps.
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Multiplex Immunohisto- chemistry（mIHC）
Common issues include signal crosstalk, undetectable partial targets, and rapid fluorescence signal attenuation, mainly affected by antibody selection, staining sequence, signal amplification conditions, and fluorochrome stability.
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Immunofluorescence (IF)
Problems like weak fluorescence signals, high background fluorescence, and damaged cell morphology tend to occur, mostly related to fluorochrome labeling efficiency, fixative selection, antibody incubation conditions, and fluorescence microscope parameter settings.
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Co-Immunoprecipitation （Co-IP）
Common issues include low precipitation efficiency, excessive non-specifically bound proteins, and undetectable target proteins, closely related to antibody conditions, lysis buffer components, incubation temperature, and washing intensity.
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Enzyme-Linked Immunosorbent Assay （ELISA）
Problems such as poor linearity of standard curves, abnormal sample detection values, and uneven color development are prone to occur, usually related to coating conditions, blocking effects, enzyme-labeled antibody concentration, and substrate reaction time.
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Flow Cytometry Assay（FCM）
Common issues include low cell sorting purity, detection signal interference, and cell clumping that clogs the pipeline, mostly related to sample preparation (e.g., single-cell suspension quality), fluorescent antibody labeling conditions, and instrument parameter calibration.
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Chromatin Immunoprecipitation Assay（ChIP）
Problems such as poor chromatin fragmentation, low antibody enrichment efficiency, and severe background DNA contamination are prone to occur, mainly affected by formaldehyde cross-linking conditions, sonication parameters, antibody specificity, and washing steps.
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