Why we use secondary antibodies (indirect detection method) - in western blots, etc. 

I haven't come across such a diligent RU-vidr before lol. Every video is really helpful! Thank you!

I have been watching your videos for a while. Thanks for making these easy-to-understand videos!! They are super helpful for my understanding my lab work and coursework!! I love your energy!

thank you!! i was curious about this because my prof didn’t touch upon why we don’t just detect primary antibodies. this was very helpful :)

I’ve always wanted to know why we use secondary antibodies! This was so easy to follow! Thank you!

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Thank you so much, I was confused about this all day and now I finally understand.

I'm planning on starting a biochemistry club in high school (rising sophomore by the way), and your vids are so helpful! thank you!!

I am relatively new to conducting Western blots. I employed the alkaline lysis method to extract whole proteins from yeast. Subsequently, I introduced the Opti Protein Marker (ABM, CAT log NO: G252) and completed the gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane using the wet transfer method. Following the transfer, I performed a Ponceau staining, during which the protein markers were clearly visible. Moving forward, I proceeded to block the membrane for 1 hour in 5% BSA in TBST (Initially, I attempted blocking with 5% non-fat skimmed milk, but encountered high background signals). Subsequent to blocking, I washed the membrane with TBST (3 x 7 mins) and TBS (1 x 5 min). Following this, I incubated the membrane overnight at 4 degrees Celsius with a primary antibody (Beta-tubulin, Rabbit IgG Polyclonal), 1:1000 dilution in 5% BSA in TBST). Afterward, I repeated the washing steps with TBST (3 x 7 mins) and TBS (1 x 5 min). For the next step, I incubated the membrane with a secondary antibody (Rabbit anti-Goat IgG (H+L), HRP, Polyclonal, 1:10,000 diluted with 5% non-fat skimmed milk in TBST) for 3 hours. Subsequently, I performed additional washes with TBST (3 x 7 mins) and TBS (1 x 5 min). Finally, I carried out chemiluminescence detection with an exposure time of 30 seconds. However, my results were unsatisfactory as I observed multiple nonspecific bands, and the protein marker disappeared. Can you please help me with this?