𝗣𝗿𝗶𝗻𝗰𝗶𝗽𝗹𝗲 𝗮𝗻𝗱 𝗧𝘆𝗽𝗲𝘀 𝗼𝗳 𝗪𝗲𝘀𝘁𝗲𝗿𝗻 𝗕𝗹𝗼𝘁 
Western blotting is allowing the detection of specific proteins in complex cell lysates. It starts with protein separation by SDS-PAGE, followed by transfer to a membrane and detection using specific antibodies [1]. There are multiple transfer techniques: 
• 𝘞𝘦𝘵 𝘵𝘳𝘢𝘯𝘴𝘧𝘦𝘳, where the gel and membrane are submerged in buffer between electrodes. 
• 𝘚𝘦𝘮𝘪-𝘥𝘳𝘺 𝘵𝘳𝘢𝘯𝘴𝘧𝘦𝘳, using minimal buffer and a pressurized system. 
• 𝘋𝘳𝘺 𝘵𝘳𝘢𝘯𝘴𝘧𝘦𝘳, commercially available as pre-assembled membranes (e.g., Invitrogen iBlot), which offer speed but sometimes lower flexibility [2]. 

𝗚𝗲𝗹 𝗣𝗿𝗲𝗽𝗮𝗿𝗮𝘁𝗶𝗼𝗻 𝗮𝗻𝗱 𝗣𝗿𝗼𝘁𝗲𝗶𝗻 𝗟𝗼𝗮𝗱𝗶𝗻𝗴 
Western blotting 𝘴𝘵𝘢𝘳𝘵𝘴 𝘸𝘪𝘵𝘩 𝘨𝘦𝘭 𝘱𝘳𝘦𝘱𝘢𝘳𝘢𝘵𝘪𝘰𝘯, either by casting your own stacking and running gels or by purchasing precast gels. Accurate protein quantification is critical to ensure detectable bands without overloading. Calculating loading amounts relative to 𝘩𝘰𝘶𝘴𝘦𝘬𝘦𝘦𝘱𝘪𝘯𝘨 𝘱𝘳𝘰𝘵𝘦𝘪𝘯𝘴 𝘭𝘪𝘬𝘦 𝘎𝘈𝘗𝘋𝘏 𝘰𝘳 β-𝘢𝘤𝘵𝘪𝘯 𝘪𝘴 𝘦𝘴𝘴𝘦𝘯𝘵𝘪𝘢𝘭 𝘧𝘰𝘳 𝘯𝘰𝘳𝘮𝘢𝘭𝘪𝘻𝘢𝘵𝘪𝘰𝘯 𝘢𝘯𝘥 𝘳𝘦𝘱𝘳𝘰𝘥𝘶𝘤𝘪𝘣𝘪𝘭𝘪𝘵𝘺 [3].  

Beware! During this workflow, small mistakes – such as displacing samples during loading, using incorrect antibody concentration, or uneven transfer- can ruin results. 

𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗮𝗻𝗱 𝗢𝗯𝘀𝗲𝗿𝘃𝗮𝘁𝗶𝗼𝗻𝘀 
Unexpected signals can appear on the membrane. These may be artifacts, antibody Sc fragments, or signs of improper transfer. Uneven bands can indicate irregular current flow, membrane contact issues, or buffer inconsistencies. Safety is critical: never touch the running gel or blotting apparatus with wet gloves. Observing the blot during transfer prevents accidents and ensures correct positioning of the gel and membrane [4].   

𝗥𝗲𝗹𝗲𝘃𝗮𝗻𝗰𝗲 𝗶𝗻 𝗜𝗺𝗺𝘂𝗻𝗼𝗹𝗼𝗴𝘆 
Western blot is invaluable in defining protein presence in immune cells, verifying expression of activation markers, cytokines, or signaling molecules. Its semi-quantitative nature provides functional validation complementary to flow cytometry or ELISA. For instance, 𝘤𝘰𝘯𝘧𝘪𝘳𝘮𝘪𝘯𝘨 𝘵𝘩𝘦 𝘦𝘹𝘱𝘳𝘦𝘴𝘴𝘪𝘰𝘯 𝘰𝘧 𝘊𝘋3ζ 𝘪𝘯 𝘊𝘈𝘙-𝘛 𝘤𝘦𝘭𝘭𝘴 or 𝘱𝘩𝘰𝘴𝘱𝘩𝘰-𝘚𝘛𝘈𝘛 𝘱𝘳𝘰𝘵𝘦𝘪𝘯𝘴 𝘪𝘯 𝘴𝘵𝘪𝘮𝘶𝘭𝘢𝘵𝘦𝘥 𝘮𝘢𝘤𝘳𝘰𝘱𝘩𝘢𝘨𝘦𝘴 ensures accurate interpretation of functional assays [5].   

Stay tuned for 𝗗𝗮𝘆 𝟯𝟴: 𝗾𝗣𝗖𝗥 𝗕𝗮𝘀𝗶𝗰𝘀 – 𝗙𝗿𝗼𝗺 𝗥𝗡𝗔 𝘁𝗼 𝗤𝘂𝗮𝗻𝘁𝗶𝗳𝗶𝗰𝗮𝘁𝗶𝗼𝗻   

References
1. DOI: 10.4103/1947-2714.100998 
2. DOI: 10.1016/j.ymeth.2005.11.007 
3. DOI:10.1021/pr1011476 
4. DOI: 10.1016/0003-2697(81)90281-5 
5. DOI: 10.1126/science.aar6711 

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