Native PAGE

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Native PAGE

Postby karthick2016 » Aug 14 2016 4:37 am

Hello guy,

I am a newbie to the forum and looking for a Native PAGE wsetern blot protocol to observe the oligomerisation of the protein. our protein pI:9.73.
we do not have sepecial setup.

if anybody willing to share the protocol it will be highly helpful.

thanks for the help.

Best
Karthick
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Re: Native PAGE

Postby mdfenko » Aug 15 2016 7:09 am

since your protein has a high pI, i recommend you use an acid native page protocol:

pH 4.3 gel (stacks at pH5, runs at pH4.3)

8X running gel buffer:
48 ml 1N KOH
17.2 ml Glacial Acetic Acid
4.0 ml TEMED (or add before pouring gel)
dw to 100 ml
pH=4.3


8X stacking gel buffer:
48 ml 1N KOH
2.87 ml Glacial Acetic Acid
0.46 ml TEMED (or add before pouring gel)
dw to 100 ml
pH=6.7


10X electrode buffer:
31.2 gm Beta-Alanine
8.0 ml Glacial Acetic Acid
dw to 1000 ml
pH=5.0

run "+" to "-"

percentage acrylamide depends on the size range of the protein mixture and the degree of separation you want. use 37.5:1 or 29:1 (acrylamide:bisacrylamide).
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Re: Native PAGE

Postby karthick2016 » Aug 15 2016 11:01 am

Hello mdfenko,

Thank you very much for the protocol and receipe. I have couple of things, i do not understand.

I guess, by '8X running buffer' you meant it as 'resolving gel'. I should use the both of stacking and resolving buffer as 1X, right?. please correct me if i am wrong.

I do not see any APS in the receipe, should i add it or ?

can you please suggest receipe for the sample loading buffer?

Thanks again,

Best
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Re: Native PAGE

Postby mdfenko » Aug 16 2016 7:10 am

yes, the buffers should be 1x in the final gel mixture (ie 1/8 of the final gel mix volume is the 8x buffer).

also, add persulfate to polymerize the resolving gel. i use riboflavin to polymerize the stacking gel.

the stacking gel will require 2 additional solutions:

stacking gel acrylamide:
10 gm acrylamide
2.5 gm acrylamide
water to 100 ml.

and

riboflavin:
4.0 mg (yes, mg not gm)
water to 100 ml
store protected from light

to prepare the stacking gel:
1 part 8x stacking gel buffer
2 parts stacking gel acrylamide
4 parts water
1 part riboflavin

after mixing, pour the stacking gel solution, place comb (if a slab gel) and expose with fluorescent light.

sample loading buffer should be just bromphenol blue in glycerol. you're not denaturing the protein so no 2-me or sds. you can add a buffer to adjust the sample pH to around 6.7 or6.8.

remember, the electrodes are reversed from "normal" page (you run "+" to "-" rather than "-" to "+".
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Re: Native PAGE

Postby karthick2016 » Aug 17 2016 10:06 am

Hello mdfenko,

Thanks again for the receipe. Still i am left with some questions.

1.What kind of light can be used to polymerise the stacking gel since you mention about the fluorescent light. could you please recommend some easily avalible fluorescent sources in a typical lab.

2. You suggested that gel run from "-" to "+", how can i acheive it ?
(for example, can i acheive by plugging pwer chord, 'red into black', and black into red of the power pack; for Bio-rad powerpack and power cord comes in red and black)

3.Since SDS and BME are not used in native PAGE, how the antigen of the protein can be umasked so that the antibody can find its antigen. usually with SDS and BME our anitbody works very nicely.

Thank you for the answers.

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Re: Native PAGE

Postby mdfenko » Aug 18 2016 7:23 am

1: a fluorescent desk lamp will work.

2: (actually, i said "+" to "-" which is the reverse of the configuration for most electrophoresis methods) that is correct, just go black to red and red to black.

3: you want to run native page to see whether or not the targeted epitope is available to the antibody under native conditions (the data sheet for most commercial antibodies tell you for which procedures the antibody will work; if it will work for elisa or ip then it should recognize the native protein). you can denature during (soak the gel in sds-bme-buffer prior to transfer and add 0.05% sds to the transfer buffer) or after the transfer by soaking the membrane in sds-bme-buffer if necessary.
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Re: Native PAGE

Postby karthick2016 » Aug 30 2016 12:33 pm

Hello mdfenko,

Thank you very much for the protcol.

i will try the protocol and get back to you for more suggestions if i struck with any problems.

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Re: Native PAGE

Postby karthick2016 » Oct 18 2016 9:59 am

Hello mdfenko,

I tried the Acidic native gel protocol ot worked but i am having problem in understanding the molecular weight, is there any protein ladder commerically avalible which i can use?

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Re: Native PAGE

Postby mdfenko » Oct 19 2016 7:29 am

hello karthick,

native page protocols separate proteins based more on net charge than size. although you can achieve some sieving by adjusting the acrylamide and crosslinker concentrations, you may still see proteins of disparate size migrating together.

i don't know of any commercial ladders for use with acid page. you may be able to prepare one yourself with a series of basic proteins. but they will still be separating based on charge.

or, you can use acid page as the 1st dimension of a 2d system (2nd dimension will be sds-page) to determine the sizes of your proteins of interest.
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Re: Native PAGE

Postby karthick2016 » Oct 20 2016 10:14 am

Hello mdfenko,

thank you for the suggestions.

Do you have any protcol for the method you suggested ''acid page as the 1st dimension of a 2d system (2nd dimension will be sds-page)'' since i do ot have any experience with the method.

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Re: Native PAGE

Postby mdfenko » Oct 20 2016 11:39 am

have you run 2d electrophoresis?

usually the 1st dimension is isoelectric focusing and the 2nd dimension is sds-page.

you can run the acid-page as the 1st dimension, cut a lane (stain the rest), soak in sds/2-me/buffer (sample buffer, you can add bromphenol blue), slide the gel slice on top of the sds-page gel (between the plates) and run as sds-page. you should use a thicker gel for the 2nd dimension to make it easier to slide the gel in.
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Re: Native PAGE

Postby karthick2016 » Nov 02 2016 9:58 am

Hello,

Thank you very much.
I never did the 2-dimensional electrophoresis.

can you please send any detailed protocol or procedure for it.

Thank you for the help.

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Re: Native PAGE

Postby mdfenko » Nov 02 2016 11:09 am

do you pour your own gels or use commercially prepared gels?
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Re: Native PAGE

Postby karthick2016 » Nov 04 2016 8:20 am

Hello,

I make my own gels for western blot.

If there is any commerical source, i am ready to buy

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Re: Native PAGE

Postby mdfenko » Nov 04 2016 6:27 pm

since you pour your own gels:

pour a non-denaturing (native) gel for your first dimension.

pour a sds gel for the second dimension.

make the sds gel thicker than the first dimension gel. for example, make the first dimension 0.75mm thick and the second dimension gel 1.0mm thick.

after running the first dimension gel, cut out a lane, soak it in sample buffer for 10-15 minutes, then slide it between the plates of the second dimension gel so that it is fully in contact with the gel. you can seal it in place with stacking gel or some agarose.
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