Hi, I have cross-linked the E-Cadherins using DTSSP. I am planning to run the western blot experiment to see if it forms higher order oligomers. I am not sure if I should use Sampling buffer and heat it for 10 minutes before loading it in the gel. Can anyone suggest if this step is necessary?
Heating the sample in SDS-PAGE sample buffer is intended to assure complete denaturation of the proteins so that they run at the correct molecular weights, so it should be done unless there is some particular reason to avoid heating your protein (some proteins seem to disappear when heated in sample buffer). What you have to be careful about, however, is that the crosslinker you used (DTSSP) contains a cleavable disulfide bond. If your sample buffer contains a reducing agent (dithiothreitol, TCEP, or 2-marcaptoethanol), the crosslinker will be cleaved and you will only see the monomer. Make sure you use a sample buffer that does not contain reducing agent. Then you can prepare one sample with reducing agent and one without. When you run the Western blot, you will be able to compare cleaved versus uncleaved, and it will be clear whether crosslinking occurred.
Very good point Adam! I believe ThermoFisher/Life Technologies NuPAGE sells 4X sample buffer without reducing agent. Reducing agent sold separately at 10X.
Hi Adam,
Today I ran the blot for cross-linked protein. The protein of interest is a fusion protein consisting of full length E-acdherin, 15 amino acid flexible linker and a fluorescent protein YFP. In one well I loaded uncross-linked E cadherin lysate and in one well the cross-linked product with concentration of cross-linker 1mM. In gel I observed two bands for both lanes. I see bands at 150kDa and at 300kDa. The dimer band for cross-linked product is weaker than uncross-linked product. I did not use reducing buffer in this experiment. However, I used sampling buffer that does not contain reducing agent. Also, I denatured the sample by heating it at 70C for ten minutes.I used DTSSP cross-linker and the cell line was HEK293T. Could you explain why I am getting strong dimer band without cross-linker and the band is there but weaker when I add cross-linker. I was expecting no dimer band in case when there is no cross-linker and strong dimer band when there is a cross-linker.
Can you show the blot? What type of gel did you run? Was the 300 kDa band in the separating gel? What conditions did you use for crosslinking? Is E-cadherin glycosylated, which would result in a higher molecular weight?
Some crosslinking of the monomers in a noncovalent dimer might occur by formation of intermolecular disulfide bonds, if there are cysteine residues close to each other and there is no reducing agent. Try preparing a reduced sample to see if the 300 kDa band disappears. The high molecular weight band could also be aggregrated protein, which probably won't respond to reducing agent.
Reaction of the protein with the crosslinker might interfere with the ability of your antibody to bind to the protein, especially if the crosslinker-to-protein stoichiometry was very high. Since you used a high crosslinker concentration, it is possible that each protein molecule is coated with many crosslinker molecules. Another possibility is that the protein is crosslinked to many other proteins, forming an aggregate that does not enter the gel and does not get transferred. Try the experiment with lower crosslinker concentrations, such as 10-100 micromolar. You may also have to try a variety of crosslinkers with different chemistries and spacer arm lengths.
Are you concerned that the fusion protein may not behave the same as native cadherin?
Hi Adam,
I am attaching the blot so that you can look at it. I used 3-8% NuPAGE tris-acetate gel. Running buffer was also tris-acetate. I followed the thermo-scientific protocol from where it is purchased. I cross-linked it at room temperature for 30 minutes. Also cross-linker was dissolved it water.
Please look at this paper:
E-cadherin functions as a cisdimer
at the cell–cell adhesive
interface in vivo published in Nature Structural and Molecular Biology in 1999 by Takeda et al
Figure 1a. They exactly did the same experiment. Used same cross-linker. Used same concentration of cross-linker as I am doing. However, there cell like was different and their protein was not a fusion protein. Also, they used different cell line. I am using HEK293T.
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