Dear Pravin

try just to dilute a fraction of your eluted protein with the same buffer containg 8M urea 1:4 (as 10ul eluted + 30ul of urea buffer) boil the sample and load it directly in SDS page. You will observe some precipitation but the gel will run properly and if you protein is not so dluted you can see it. Guadinine precipitation in my experience do not affect the SDS-page when the concentration is lower that 2M.

good luck

Manuele

Make sure that the samples in the adjacent lanes have the same amount of guanidine, otherwise your sample will spread to the sides while running through the gel..

We have described an ethanol precipitating step, as in Article Tau Proteins in the Temporal and Frontal Cortices in Patient...

Hope it helps.

Elizabeta B Mukaetova-Ladinska yes i tried

1 TCA precipitation

2 Ethanol precipitation

3 dilution of GuHCL to 2 M with Urea and Water(buffer)

by Ethanol and TCA protein is not precipitated

when i diluted protein to 2M GuHCl and added sample buffer (SDS+DTT + blue color dye + tris buffer) samething is precipitating out from solution

* this sample buffer i am using from invitrogen.

* my interest of protein is membrane protein of size 15KDa

did you try diiluting the sample - maybe the protein concentration is too high? Also, sonicating the sample?

Elizabeta B Mukaetova-Ladinska

i tried dilution of GuHCl upto 1M including sample buffer.

for dilution i use water or 8M urea, but this dilution is not worked for my case.

Irina Boksha

Manuele Martinelli

Hi Manuele, what about then you have Laemelli buffer without b-ME?

We have a protein we introduced a Cysteine mutation and dimerized it under oxidating conditions. We want to run the SDS-PAGE get to make sure it is at the right MW, but without breaking the disulfide bond. We have the protein in 5M guan and dilute 1/3 in H20 or PBS1x, but this does not seem to work well. The protein crashes out of solution to the point we cannot load it on the gel.

Dear Norelle C. Wildburger

You can load protein with out reducing in SDS-page.

I think that in your specific case the problem is the protein precipitation that happen when you decrease the guanidine.

Try to dilute the sample at leat 4 times in Urea 6M instead PBS and then mix with laemil with and with-out BME and load it again in SDS-page. Do not worry if you see some precipitate because is it possible that this is not the protein but the precipitation of the guanidine-sds complex.

good luck

Manuele

Manuele Martinelli

Ok thanks will do. But what about Heat? we use 95C for normal PAGE, but in this special case without B-ME we use 85C for 5 min.

Dear Norelle C. Wildburger

It depends a lot from the nature of your protein

Is it theoretically a soluble protein but it is expressed in inclusion bodies or is it a membrane protein?

For soluble proteins generally i'm using the standard protocol (95°C) with and with-out reducing. The only exception are the mabs for which in abscence of reducing, I incubate 1' at 95°C instead 5' because i found that 5' incubation at 95°C destabilize the mabs and it seem that are degraded also if it is not.

For membrane proteins (but my experience in this field is very limited) i kwon that it is better reduce the temperature (also at 50-60°C) because the heat can induce aggregation and the protein may not run properly on the gel.

However in some cases the best think to do, is try different conditions and compare the results because not all the proteins are the same.

good luck

Manuele

Manuele Martinelli

The protien is a soluble monomer, but we have introduced a S>C point mutation and are making dimers under oxidating conditions. In protparam the dimer Grand average of hydropathicity (GRAVY): -0.379

Can we run native gel instead of sds gel for the samples containing guanidine hydrochloride?

I have recently trouble shooted it.

Dillute ur sample with 1x running buffer and then add 5X loading dye with bme and heat at 100°C for 10 min.

Pre heat ur running buffer at 70°C add to the tank load sample keep voltage @110 let it stack inside a incubator maintained @ 50°C after the samples are stacked it out run it as usual.. Or u can just simple TCA precipitate ur samples first the dissolve in an appropriate buffer and then run

(answer I posted somewhere else already, sorry)

The trick i've been using for a long time is to put a couple of uL of concentrated sample in 6M GdnCl in 5 to 10X the volume of pure SDS loading buffer, without boiling. It then causes a significant part of the guanidine to precipitate with the SDS (almost instantly). I then centrifuge and directly load the thereby strongly desalted supernatant in the gel.

for running the gel, I recommend using low voltage/amperage, especially in the beginning of the migration. that should take care of the remaining distortions of the migration due to remaining high salinity.

sample preparation barely takes one or two minutes more than for a normal set of samples, running the gel about 50% more time.

for a result, check out figure S1 of this paper (made from samples with 6M GdnCl)

Article Site-Specific Studies of Nucleosome Interactions by Solid-State NMR