Using Leammli buffer and heat treatment prior to the loading step already fully denatures the IgG. Do not need to use urea additionally. General sample prep for SDS-PAGE presents a denatured and reduced form of antibodies as heavy and light chain bands.

Hi Mariette Bonnet, like İsmail Emir Akyildiz answered, it is more easy denatures IgG using Leammli buffer and heat, it is a more effective and straightforward method than urea denaturation.

Hi there,

8M urea is the maximum you can dissolve in the loading buffer. I use such loading buffer to solubilize proteins from beads in CoIPs. Its composition is : 8M urea, 5%SDS, 40mM Tris-HCl pH6.8, 0.1 mM EDTA, 1% BME, 0.04%BB. The only issue is to make sure every well of the gel is loaded with equivalent amount of this buffer otherwise urea tends to diffuse lateraly during gel running.

Thank you both for your answer!

We have tried heating up the sample with Laemmli buffer, but got a smear between 150 kDa and 60 kDa on silver-stained gel.

it looked like fragmentation (see gel attached,lane 5-12 and 14-15 heated sample at 95°C for about 2min30, lane 13 same as 12 but not heated). we may have not heated the sample long enough to get sharp band.

We actually want to estimate the amount of impurities after purification. The band we are looking at is around 65 kDa, and is about 1000 times less abundant than IgG.

any input on how to proceed would be greatly appreciated!

Dominique, thanks for your answer.

so the recipe you give would be a 2x loading buffer, right?

or did you add your buffer directly to your beads?

Hi again,

I use it directly on beads but you may use it as a x1.33 LB (1 volume of sample + 3 volumes of LB) as long as urea concentration remains at least at 6M to ensure its denaturant/chaotropic effect on proteins.

Thanks a lot!

we will try that :-)

Hi again,

I would suggest to load less material into wells in order to sharpen the bands in the protein pattern.

thanks for your input.

We cannot really load less protein. We want to estimate the amount of an impurity at about 50kDa.

If we decrease the protein amount loaded, we might not see it anymore

Hi again, sample overloading results in smearing and unresolved bands...