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acquity 03-26-2007 07:52 PM

Optimum working concentration in SDS-PAGE?
 
Hello everybody,
I have just started working with SDS-PAGE and still trying to learn it.
Is there an optimum working concentration for loading samples.
Of course staining protocol is equally important, I know this point.
but is there an upper limit and lower limit for loading samples?

I will be grateful if anyone can help.

moleculardude 03-26-2007 08:43 PM

Re: Optimum working concentration in SDS-PAGE?
 
Hello Acquity!

There are a few rules about loading protein samples on SDS-PAGE gels.

The first is that if you overload the gel with excess protein, you risk streaking of blots. Also if you expect to see differences in the lanes, this may be a problem as the blots easy saturate upon ECL development with excess protein (large bands very dark for all).

SO here are some loading rules:

1) The amount of protein you load depends on the well size and gel thickness. It has been noted that if you load 10 µl of a 2 mg/ml final concentration of denatured protein per sample well, you get better gel usually.

2) In general, the amount of protein sample you should load onto your SDS-PAGE gel should range from 0.5–4.0 µg for purified (recombinant) samples to about 40–60 µg for crude samples (this includes Coomassie blue staining)

Silver staining methods are about 100-fold more sensitiveand thus require less protein per sample.

3) Working concentrations of proteins should all be equal throughout the gel to prevent irregularities in running (due to differences in the gel salt etc) and most importantly to be able to compare different treatments / conditions (different lanes).

If anyone has more information or tips, please contribute them here!

thanks :)

acquity 03-27-2007 09:39 AM

Re: Optimum working concentration in SDS-PAGE?
 
Quote:

Originally Posted by moleculardude (Post 2934)
Hello Acquity!

There are a few rules about loading protein samples on SDS-PAGE gels.

The first is that if you overload the gel with excess protein, you risk streaking of blots. Also if you expect to see differences in the lanes, this may be a problem as the blots easy saturate upon ECL development with excess protein (large bands very dark for all).

SO here are some loading rules:

1) The amount of protein you load depends on the well size and gel thickness. It has been noted that if you load 10 µl of a 2 mg/ml final concentration of denatured protein per sample well, you get better gel usually.

2) In general, the amount of protein sample you should load onto your SDS-PAGE gel should range from 0.5–4.0 µg for purified (recombinant) samples to about 40–60 µg for crude samples (this includes Coomassie blue staining)

Silver staining methods are about 100-fold more sensitiveand thus require less protein per sample.

3) Working concentrations of proteins should all be equal throughout the gel to prevent irregularities in running (due to differences in the gel salt etc) and most importantly to be able to compare different treatments / conditions (different lanes).

If anyone has more information or tips, please contribute them here!

thanks :)



Thank you very much for your answer it will be very helpful for me

WBO 08-14-2010 02:30 AM

Re: Optimum working concentration in SDS-PAGE?
 
Hello all,
When I run my gel,my samples spills in the solution and disturb the lane( mixed).Can I load my protein before filling the running buffer to the apparatus?My protein is also not develop any signal for western blot but develop the band for coomasse blue and silver stain.I am using 10% gel and load about 30 micrometer of purified sample.Any suggestion is appreciated
Thank you
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nisha sinha 06-03-2012 10:50 AM

Re: Optimum working concentration in SDS-PAGE?
 
Even me facing the same problem.plz help....

butters 06-04-2012 04:46 AM

Re: Optimum working concentration in SDS-PAGE?
 
Nisha, could you be more specific. How much you are loading and what is the capacity of the wells.

Do provide your formula for the loading buffer

nisha sinha 06-04-2012 05:17 AM

Re: Optimum working concentration in SDS-PAGE?
 
I have loaded 15ul of my sample and 5ul 1x sample buffer (with DTT) and did silver staining. I can see the bands and also got the profile i wanted.

Depending on this i decided to run blot for CD63. It is recommended to use non reducing conditions.

My isolation of the sample requires DTT at a concentration of 200mg/ml so have used DTT for isolation.But i ran the gel in non reducing conditions ( sample buffer without DTT ) since it is recommended.

DTT will hinder protein estimation by BCA so i randomly loaded crude sample 15 ul of sample and 7.5ul of 2x sample buffer without DTT.
But i can't see any bands.

My positive control for CD63 showed bands.Its a cell line so lysate was prepared using 100mM DTT in lysis buffer.

Also plz let me know if acetone precipitation can be used for removal of interferring substances i.e. DTT and if yes please share the protocol.

butters 06-04-2012 09:06 AM

Re: Optimum working concentration in SDS-PAGE?
 
I am confuse regarding your detail. You couldn't see band in your blot or PAGE (silver staining?)?

Sorry for DTT removal I do not possess any protocol. Have you ask your labmate? Since most likely they would have the protocol.


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