Western Blot Analysis Steps: A Comprehensive Guide

Western blot analysis is a crucial technique in molecular biology used to detect specific proteins in a sample. The process involves several steps, each designed to ensure the quality and accuracy of the results. In this article, we will delve into the western blot analysis steps, highlighting the importance of quality control measures and the various techniques employed to guarantee high-quality outcomes.

Recovery and Dilution Linearity

The first step in western blot analysis is to determine recovery, which is typically measured between 80-130% (with a maximum tolerance of 70-150%). To achieve this, biological samples are spiked with varying levels of target protein. Additionally, dilution linearity is tested by performing 2-fold and 4-fold dilutions of the biological samples.

Lot-to-Lot Consistency

To ensure consistency between lots, RayBiotech compares calculated concentrations of 2-3 positive control samples with calibration curves from current and previous lots (with a maximum tolerance of 20% CV). This step ensures that the results are reliable and comparable across different batches of the product.

Inter-Assay Reproducibility

To evaluate inter-assay reproducibility, at least two independent experiments are performed using 2-3 positive control samples and a full standard curve (with a maximum tolerance of 12% CV). This step helps to validate the results and ensure consistency across different assays.

Intra-Assay Reproducibility

For sandwich ELISA kits, intra-assay reproducibility is evaluated by running 2-3 positive control samples in duplicate on a single plate (with a maximum tolerance of 10% CV).

Product-Specific Quality Control Tests

At RayBiotech, we take our product quality very seriously. Every kit produced is subjected to a rigorous battery of product-specific quality control tests and manufactured in compliance with GMP and ISO 13485 standards.

Recommended Dilution Ranges for Serum and Plasma

The biological samples tested include serum, EDTA plasma, citrate plasma, heparin plasma (from normal healthy donors), and cell culture medium (DMEM or 1640). Our recommended dilution ranges are determined by testing these samples using our western blot analysis kits.

In conclusion, western blot analysis is a powerful technique that requires careful attention to quality control measures. By following the steps outlined above, researchers can ensure reliable and accurate results in their experiments. At RayBiotech, we are committed to providing high-quality products and services, and we believe that our commitment to quality control sets us apart from other companies in the industry.

Western blot analysis steps