Sure Brindaban. Here is a detailed explanation: "A negative control lysate is a lysate from a sample known to not express the target protein. This control is important for determining whether non-specific binding (false positive result) has occurred in the western blotting procedure. Commonly used negative controls: • Samples from knockdown or knockout tissue/cell lines • Samples from RNA interference targeted lines • Cell line/tissue/experimental condition with proven negative signal". For more information, check this guide here: images.novusbio.com/design/BR_westernblotguide_042816b.pdf
Thanks for making such an easy-to-understand video on IP and Co-IP. Could you kindly suggest a book where I can learn more about how to explain the Co-Ip results on graph?
Patricia, FLAG is an 8-amino acid tag attached to a protein, usually for its easy purification. You can find more info here: en.wikipedia.org/wiki/FLAG-tag
Sure Ranjit. I use Protein Data Bank (PDB) to get the crystal structures and use Pymol and UCSF Chimera to make the images. All three are free to use, and you can find a lot of free tutorials on UA-cam about making these images. Good luck!
Sir, suppose that by doing Co-IP , we have found out that three proteins are interacting with my target protein by doing Mass Spectrometry. Now, how can l separate my target protein from other three interacting proteins??
For that, you can use TAP-tagging technique. It keeps the proteins in active state. Take a look here for more details: en.wikipedia.org/wiki/Tandem_affinity_purification, and www.nature.com/articles/nbt1099_1030
And you can also use SDS-PAGE to separate the proteins of complex. But of course, it will denature them. Once you have made sure the identity of interacting proteins, you can clone and express them on their own to study them further.
@@SpartanTutorials if we use SDS-PAGE, the polypeptides of the Proteins will get separated according to their molecular weight.....so how can l discern which polypeptide chain belongs to my target protein?
@@ranjitshaw9110 You're right. Here, we will use the information from Mass Spec. Mass Spec has already told you hopefully which proteins are these. If you are working with a model organism whose genome is already sequenced (very likely), then we can just go to NCBI and see what's the predicted mol. wt. of that protein. The two (predicted and SDS-PAGE) should match.
![Lp. Сердце Вселенной #60 РОЖДЕНИЕ ЛОЛОЛОШКИ [Финал] • Майнкрафт](http://i.ytimg.com/vi/YoR0pAV9FVQ/mqdefault.jpg)
nice explanation
Sir, can you please elaborate a little bit on negative control during WB?
Sure Brindaban. Here is a detailed explanation: "A negative control lysate is a lysate from a sample known to not express the target protein. This control is important for determining whether non-specific binding (false positive result) has occurred in the western blotting procedure. Commonly used negative controls:
• Samples from knockdown or knockout tissue/cell lines
• Samples from RNA interference targeted lines
• Cell line/tissue/experimental condition with proven negative signal". For more information, check this guide here: images.novusbio.com/design/BR_westernblotguide_042816b.pdf
Thanks for making such an easy-to-understand video on IP and Co-IP. Could you kindly suggest a book where I can learn more about how to explain the Co-Ip results on graph?
Thanks Saiful. For your analysis, you may find this paper helpful: www.ncbi.nlm.nih.gov/pmc/articles/PMC3008648/pdf/btq652.pdf
Excellent explanation. Thanks alot sir
Most welcome Sushmitha.
I am having to analyze a figure for IP and on the left is has IP and FLag can you explain what the FLag is
Patricia, FLAG is an 8-amino acid tag attached to a protein, usually for its easy purification. You can find more info here: en.wikipedia.org/wiki/FLAG-tag
Where can l get the beautiful pictures of the proteins which you showed here?
Can you please tell me sir!!
Sure Ranjit. I use Protein Data Bank (PDB) to get the crystal structures and use Pymol and UCSF Chimera to make the images. All three are free to use, and you can find a lot of free tutorials on UA-cam about making these images. Good luck!
@@SpartanTutorials okay, Thankyou so much sir for these valuable informations
@@ranjitshaw9110 Most Welcome!
I have some questions about the immunoprecipitation and western blot results plz how can I ask you?
You can ask on our Android App. Download it from the Play Store. Would be happy to answer your questions.
Sir, suppose that by doing Co-IP , we have found out that three proteins are interacting with my target protein by doing Mass Spectrometry.
Now, how can l separate my target protein from other three interacting proteins??
For that, you can use TAP-tagging technique. It keeps the proteins in active state. Take a look here for more details: en.wikipedia.org/wiki/Tandem_affinity_purification, and www.nature.com/articles/nbt1099_1030
And you can also use SDS-PAGE to separate the proteins of complex. But of course, it will denature them. Once you have made sure the identity of interacting proteins, you can clone and express them on their own to study them further.
@@SpartanTutorials if we use SDS-PAGE, the polypeptides of the Proteins will get separated according to their molecular weight.....so how can l discern which polypeptide chain belongs to my target protein?
@@ranjitshaw9110 You're right. Here, we will use the information from Mass Spec. Mass Spec has already told you hopefully which proteins are these. If you are working with a model organism whose genome is already sequenced (very likely), then we can just go to NCBI and see what's the predicted mol. wt. of that protein. The two (predicted and SDS-PAGE) should match.
@@SpartanTutorials ok sir understood now... Thankyou for the clarification