Basic guide to mastering In-Cell Western assays
Use this basic guide to aid you in troubleshooting the common issues encountered during in-cell Western assays. Whether you are a seasoned investigator or just beginning to explore the world of ICW, this is a resource that will equip you with the knowledge and strategies you need to successfully navigate unique intricacies.
Common issues with In-cell Westerns
In-cell Westerns (ICW) assays are powerful and versatile tools in the arsenal of cell biologists and protein research. These assays offer the unique advantage of quantifying target proteins and assessing protein expression and activation directly within the native cellular context, providing valuable insights into cellular processes, signaling pathways, and treatment responses.
Like any experimental technique, ICWs are not immune to challenges and pitfalls. From poor antibody specificity and high background to nuances in staining, imaging platforms and quantification, these challenges can hinder progress and data interpretation.
Pipette Carefully
A great rule of thumb to follow when working with cells is to never touch the bottom of the
ICW plate (or any plate for that matter) with the tip of the pipette. Touching the bottom of the plate causes defects and prevent the cells from adhering properly.
Cell Quality and Health
Poor cell health can lead to unreliable results.
Determine Linear Range
For quantitative results, the number of cells plated and the concentration of primary antibody both need to be optimized prior to testing. In the case below, plating 100,000 cells per well resulted in inconsistencies since the cells were too confluent and began to peel off of the surface of the plate along the edge of the wells. Maximum linearity was observed when 1563-25,000 cells/well were used. This antibody showed good linearity at all dilutions used during optimization, so a 1:200 dilution is suitable to conserve precious and expensive antibody.
Fixation and Permeabilization
Inadequate fixation and permeabilization can result in incomplete antibody penetration and uneven staining.
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Antibody specificity
Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity
Blocking
Insufficient blocking can lead to high background signals.
Primary Antibody
Your choice of primary antibodies matters. Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity. Be careful: using low quality primary antibodies can yield weak or nonspecific signals.
How to select the best primaries to use when dealing with in-cell Westerns:
Secondary Antibody
Just like with primary antibodies, your choice of secondary antibodies matters too. If you use inappropriate or cross-reactive secondary antibodies, it can result in nonspecific staining.
Three tips for using appropriate secondary antibodies that provide quality results:
Incubation
Variations in incubation conditions can affect staining consistency. Overnight incubation times can lead to increased background.
Washing
Inadequate washing can leave residual unbound antibodies, leading to high background.
Imaging
Using improper imaging settings or imagers with high crosstalk can result in inaccurate data.
Use lasers imagers with desired fluorophores for crosstalk in the proper wavelengths.
The Sapphire FL is capable of imaging in-cell Westerns very well, due to its sensitivity and speed. With four fluorescent channels (including two NIR channels), the Sapphire FL facilitates multiplex detection of blots and of 96-well plates with the AzureCyto In-cell Western Kit like a pro.
Test fluorophores and antibody pairs to determine the correct imaging settings for linearity.
Use consistent settings for capturing images and quantifying signals.
Normalization
Choosing the wrong normalization method can lead to misinterpretation of results.
Replicates and Controls
Inadequate replicates or lack of appropriate controls can compromise data quality.
Using an in-cell Western kit such as the AzureCyto In-Cell Western Kit can cut down on the time spent troubleshooting and optimizing your assay. The AzureCyto In-Cell Western Kit enhances the consistency and reliability of results by providing researchers with validated reagents, standardized protocols, and quality controlled components reducing experimental variability and ensuring accurate and reproducible data.
