Isoelectric focusing gels II Protein Electrophoresis

Isoelectric focusing (IEF) is one of the most commonly used techniques for the separation of proteins. IEF separations are based on the pH dependence of the electrophoretic mobilities of the protein molecules. Isoelectric focusing makes use of electrical charge properties of molecules to focus them in defined zones in a separation medium. It is the focusing mechanism that distinguishes IEF from other separation processes and makes it unique among the separation methods. In sharp contrast, the basic separation mechanism of IEF imposes forces on the molecules that directly counteract the dispersive effects of diffusion. During the separation process, the molecules in the sample accumulate in specific and predictable locations in the medium, regardless of their initial distribution. This focusing mechanism also distinguishes IEF from various modes of electrophoresis. It is important to note that IEF is a high-resolution method. It is well suited for both analytical and preparative applications.
GENERATION OF pH GRADIENTS
IEF is a very useful practical technique where simple methods for establishing and maintaining pH gradients are available. The pH gradient is essential for this technique, and the nature of the pH gradient largely determines the quality and usefulness of the separation. The two most widely adopted methods for generating pH gradients make use of different types of synthetic buffering molecules (a) Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. They possess both acidic and basic functional groups and form pH gradients under the influence of electric fields. (b) Synthetic buffer compounds containing reactive double bonds, called acrylamide buffers, can be incorporated into polyacrylamide gel matrices. When used in the correct proportions, acryl- amido buffers generate immobilized pH gradients under the influence of electric fields.
PROTEIN DETECTION AND IMAGING
Gel-based IEF has a strong worldwide user base that supports a commercial pipeline of instrumentation as well as consumable products and will thus certainly remain a relatively low-cost, routine laboratory technique in the coming years. These techniques involve the use of many of the same dyes and stains that were developed for polyacrylamide electrophoresis gels but have been adapted for use with IEF gels.

[@chintansingh5023]

You are awesome ... Thanks to explaning in such a easy manner... Thanks a lot❤️

[@BioMagica]

😊😊😊

[@rakshithm1257]

Awesome ,I have a test on Electrophoretic techniques tomorrow, Thank you for clearing the concept

[@BioMagica]

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[@BioMagica]

Refer link for updated video without music: th-cam.com/video/iGlPtSx-Fe0/w-d-xo.html

[@christophergoudarzi9992]

Very helpful, thank you!

[@BioMagica]

Happy to help ☺️

[@anastasyamelnikova652]

great video and explanation!

[@BioMagica]

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[@mridulasoni9335]

Superb

[@BioMagica]

Thankyou 😊

[@prernasingh4660]

Thank you so much for making this video

[@BioMagica]

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[@dhanyamariejos3847]

Clearly understood..Many thanks!!

[@BioMagica]

😊😊😊

[@ayushijaiswal9792]

Well explained

[@BioMagica]

Thankyou

[@iqbalhussain2568]

Thank you. High informative

[@BioMagica]

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[@Samadpakara]

Nice explanation thank you

[@BioMagica]

😊😊😊

[@ioannaprasopoulou9016]

Thank you for your video :)

[@BioMagica]

😊😊

[@SeemaRani-ji2ee]

Very helpful 🙃thanks🙃

[@BioMagica]

Happy to help 😊😊😊

[@ankithanayak225]

Nice ❤️❤️

[@BioMagica]

Thankyou

[@shahidkhanalvi9519]

Thank you ❤️

[@BioMagica]

Welcome 😊😄

[@anantnalkul9898]

Just amazing...

[@BioMagica]

Happy to help 😊😊😊. Stay safe

[@debolina9090]

thank you so much..it was highly informative

[@BioMagica]

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[@priyadarshinipadhi2081]

nicely explained. thanks ma'am.

[@BioMagica]

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[@nishavijayasarathi101]

It was very helpful

[@BioMagica]

Thanks 😊

[@greciai.riverarodriguez5601]

Thank you!

[@BioMagica]

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[@ykl178]

Great explanation thank you.

[@BioMagica]

Welcome 😄 do share 😇

[@Sainan784]

Thanks 🙏

[@BioMagica]

😊😊😊

[@annaaditi126]

Clear, lucid explanation👍🏻

[@BioMagica]

Thankyou dear....do share

[@AshishrAJ-fi8cq]

thank you soo much

[@BioMagica]

You're welcome!

[@glorious7072]

Awesome

[@BioMagica]

Happy to help 😊😊😊

[@nirbhaytomar2045]

Thank u mam 😊

[@BioMagica]

Happy to help 😊😊😊

[@nancymalik3912]

Love u mam for this wonderful explanation

[@BioMagica]

Thanks 😊😊😊

[@mazbowo1403]

Thanks.... :)

[@BioMagica]

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[@shivaniathani8082]

Very helpful 👍🏽

[@BioMagica]

Thank-you. Do share 😇

[@satabdeepanda6810]

Thank uuu so much ma'am 😊😊

[@BioMagica]

Happy to help 😊😊😊

[@kulsoomjaved1164]

Very much helpful ❤️

[@BioMagica]

Glad you think so!

[@rehanabintirasool9700]

Thanks a tonnes😃

[@BioMagica]

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[@ayesharoshanara9541]

Tanq mam for such a vedio

[@BioMagica]

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[@kashishgupta5443]

awesome

[@BioMagica]

Happy to help 😊

[@maisa3eed334]

🥰🥰🥰🥰🥰🥰

[@BioMagica]

thankyou

[@sarahkivo81]

why does the positive charge decrease on the molecule as it moves towards the cathode?

[@BioMagica]

The charge is changing due to the pH gradient we created ( due to change in pH)

[@KABSOOLH1]

what does the music ha to do with the PI? i dont understand explain plz

[@BioMagica]

Music ?

[@GK-lo4ef]

thnk yu

[@BioMagica]

Welcome

[@RohitSaini-kc8xv]

Computation method

[@siratpannu5820]

Mam how can we know that a single band at PI has a single band or multiple band ?

[@BioMagica]

U mean how to know there are more than one protein in one band? Well for that u can do other techniques like western blot

[@siratpannu5820]

Okay mam 😊thnku so much

[@BioMagica]

Happy to help. Stay safe 😊

[@piyushghoshe2581]

How pH Gradient over a gel is maintained at different level ?

[@BioMagica]

In electric fields, carrier ampholytes partition into smooth pH gradients that increase linearly from the anode to the cathode. The slope of a pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes.
After the pH gradient is created, no further separation can happen in the electric field, which maintains the pH gradient. Then you apply your sample and now only only your protein sample will move according to it's pI value.

[@piyushghoshe2581]

Thank you

[@zaara8151]

Video was so informative thnk you so much..... music was soo irritating no need for any king of dumb music 🙄🙄🙄

[@BioMagica]

yes got it. thaNKYOU

[@nandulamatamsowmya86]

Sis please remove background music

[@BioMagica]

Yes I understand it is annoying. I kept it down for future videos. Will reupload these videos without the music asap

[@soubhikdas8130]

Reading wilson walker..still wrong reading

[@BioMagica]

What is it that you did not get?

[@shwetanidhi]

:)

[@BioMagica]

Why sad

[@shwetanidhi]

No no...posted by mistake....actually you saved me :)

[@poojamishra5428]

Not satisfied 😐

[@BioMagica]

What was missing ?

[@chanchaltyagi1206]

Chanchal tyagi

[@BioMagica]

OMG. You started this, and yet BINOD got famous. sorry bro

[@harshagautam5986]

Music is so distracting ! Can't watch at all

[@BioMagica]

i reuploaded this video without the music. New video link is pinned on the comment section. Sorry.