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

the bumbling biochemist
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29 окт 2024

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Комментарии : 17   
@andykim4068
@andykim4068 Год назад
I haven't come across such a diligent RU-vidr before lol. Every video is really helpful! Thank you!
@thebumblingbiochemist
@thebumblingbiochemist Год назад
Thanks so much! It really means a lot to me to hear I'm helping people
@jillianwu8610
@jillianwu8610 Год назад
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!
@thebumblingbiochemist
@thebumblingbiochemist Год назад
That's really nice to hear 😊 Thank you! I'm so happy you find them helpful and wish you all the best of luck with your work!
@noteinna
@noteinna Год назад
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 :)
@Fluor488
@Fluor488 Год назад
I’ve always wanted to know why we use secondary antibodies! This was so easy to follow! Thank you!
@thebumblingbiochemist
@thebumblingbiochemist Год назад
so great to hear!! thank you!
@brandyb9527
@brandyb9527 5 месяцев назад
Your videos are so helpful! I always have questions long after the day is done and get stuck. So thank you very much!!! ✨🙂
@thebumblingbiochemist
@thebumblingbiochemist 5 месяцев назад
I'm so glad I could help! Good luck!
@ray47891
@ray47891 Год назад
Thank you so much, I was confused about this all day and now I finally understand.
@thebumblingbiochemist
@thebumblingbiochemist Год назад
I'm so happy to hear I could help!
@asetix4709
@asetix4709 Год назад
I'm planning on starting a biochemistry club in high school (rising sophomore by the way), and your vids are so helpful! thank you!!
@thebumblingbiochemist
@thebumblingbiochemist Год назад
That's awesome! It's great to get exposure to biochemistry so early! Very glad I could be of help!
@ScientySundar
@ScientySundar 10 месяцев назад
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?
@thebumblingbiochemist
@thebumblingbiochemist 10 месяцев назад
If by marker you mean ladder, that wouldn't be expected to show. And your antibodies might just not be very specific
@ScientySundar
@ScientySundar 10 месяцев назад
@@thebumblingbiochemist Means what, we can't able to see protein ladder at the end of chemiluminescence detection? I am just confused. Can you please provide any sources or references?
@thebumblingbiochemist
@thebumblingbiochemist 10 месяцев назад
correct - you'll only see what the antibodies bind to. google has lots of info - hope you find what you're looking for
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