I'm not sure exactly what you're asking, are you trying to measure protein concentration or are you just having issues with MW determination via SDS-PAGE? I'm a big advocate of the freeze-thaw and/or french press method of cellular lysing before I add SDS sample buffer. If the issue is with your SDS-PAGE I would look at the type of buffer you're using. Personally I make mine up as 4X with the following:

2.5 mL 1M Tris-HCl pH~6.8

0.5mL ddH2O

1g SDS

0.8 mL 0.1% Bromophenol Blue

4 mL 100% Glycerol

2 mL B-Mercaptoethanol 100%

Adjust final volume to 10 (it should be already) with ddH2O

If you're having serious issues with protein aggregation you can alternatively up the glycerol content by making the 10X Variant:

a few mLs of 70:30 Glycerol:ddH2O and dissolve the solids into that:

0.606g TRIS-Base

2.5g SDS

Adjust pH to ~6.8 (strips)

2mg Bromophenol Blue (not as soluble in glycerol, but it can go in)

Adjust to 10mL w/ 70:30 (Store at -20C)

& Add B-Mercapto just before denaturation.

Dear Vincent,

Thank you for your answer.

My issue is that apparently when you cross-link proteins with glutaraldehyde and paraformaldehyde you cannot reverse the cross-linking with SDS and beta-mercaptoethanol/DTT. In the literature I can find both answers and was just wondering if someone has experience on this.

We prepare our own loading buffer (5x). The protocol I use is the following:

- 2g SDS

- 6.5 ml 1M Tris pH 6.8

- 5.8 ml 85% glycerol OR 4.93 ml 100% glycerol

- 1.55g DTT

- 6.7 ml MilliQ-H2O

- a spatula tip of Bromophenolblue

/Nuno

Dear Nuno

Glutaraldehyde and formaldehyde can react with the primary amine functional groups on proteins, such as the side chain group of lysine and amino-termini of proteins. The cross-linking reaction (depicted in attached file) as a result of glutaraldehyde treatment forms an Schiff base covalent linkage that is irreversible.

The cross-linking consequence definitely affects the accurate quantification by using SDS-PAGE, because the molecular weight of the target protein will get twice or bigger than the theoretical one and results in heterogeneity in the bulk sample.

Even thought I don’t know why you’d like to open the linkage again after the cross-linking reaction, that can be achieved by using the ethylene glycol bis(succinimidyl succinate) cross-linkers (EGS), instead of glutaraldehyde. EGS linkage can be cleaved again at pH 8.5 using hydroxylamine for 3 to 6 hrs. at 37°C.

For your reference: Volume 87, Issue 3, 13 April 1979, Pages 734-742 Biochemical and Biophysical Research Communications.

Thank you Yun-Tzai for your tip but I have made already the cross-link with glutaraldehyde so I cannot use the linker you suggested.