My name is Maria and I'm working at Y Healthcare in NOS in Sweden. Cell surface proteins are often involved in communication between cells and their environment and are therefore particular interest in research, aiming to gain more insight to diseases as well as other biological variations induced by changes in the cell environment. In this video I'm going to show you a simple side-eye dodge cell surface labeling protocol and you will be able to visually enrich these low abundant proteins without physical enhancement and study the differences of these cell surface proteins with the technology.
If you find this technology interesting, you can find more information in the 2D handbook. Moreover, we have an application note on this particular topic which you can download from job In the cell surface labeling protocol seen on top, you label the cells while they are still intact. During the labeling process, the dye will only have access to the cell surface proteins.
After the labeling step, the cells are analyzed to verify cell surface specific labeling. The label sample was fractionated into membrane and cytosolic proteins. A non fractionated sample was prepared in parallel.
For comparison, this fractionation analysis is not necessary, but was done here just to show that the cell surface protocol is specific for cell surface pro tips. We also performed the standard ETH non diet labeling protocol seen below the cells are lies before labeling, and in this way all cellular proteins are accessible for labeling. After the labeling step, the samples are subjected to 2D electrophoresis.
A adherent cells are detached using a non enzymatic procedure to avoid digestion of the cell surface proteins targeted. In this protocol, we use the rubber policeman, but using enzyme three cell dissociation media is also an option count and divide the cell suspensions into quas of five to 10 million cells per tube. The cells are then pelleted and washed in H-P-S-S-P-H 7.4 to remove traces of cell culture.
Media contamination from serum proteins and fluorescent components can interfere with labeling and detection. Cells growing in suspension are directly pelleted and washed before the labeling step after the wash. The cell palette is resuspended in 200 microliters ice cold labeling buffer containing HPSS pH 8.5 and one molar uria.
For optimal labeling conditions of cell surface proteins always check pH. Before labeling, we used 600 pic mold side-eye for 10 million CHO cells. The optimal ratio of side-eye to cell number will vary depending on the cell type.
Since we do not know the exact protein concentrations on the cell surface. How to determine the optimal conditions for side-eye labeling of proteins is described in the 2D electrophoresis principles and methods handbook. The cells are incubated with side eye D floor, minimal dice for 20 minutes on ice in the dark.
After the labeling reaction, the unreactive dye is quenched by adding 20 microliters of 10 millimolar lysine. The label cells are now washed twice in cold HPSS pH 7.4 buffer to remove the excess side eye. There will therefore be no free dye left for unwanted intracellular labeling or proteins in the next step, which is cell lysis.
The proteins on the cell surface are now labeled and the cells are washed and ready to be liced. The pellet from the lost washing step is resuspended in 150 microliter cold lysis buffer containing seven molar uria, two molar thi uria, 4%chaps 30 millimolar trees five millimolar magnesium acetate pH 8.5 and left on ice for at least one hour with occasional vortexing taxing. The samples are now ready for 2D separation.
The first step into the electrolysis is to prepare IPD strips for rehydration. Prepare district rehydration solution by adding IPG buffer corresponding to the pH interval of the strips used, and add the solution in the lens of the rehydration tray. Remove the protective film of the IPG strip and place the strips carefully with a dried gel facing down in the rehydration tray containing the rehydration solution.
Close the lid of the IPG box and rehydrate the strips overnight. In the first dimension, isoelectric focusing, the proteins are separated according to their pi. This is performed using the IPG four three.
The rehydrated strips is placed in the manifold and the electrode is mounted on top. 50 micrograms from each sample were applied using sample application caps. We have either directly applied non fractionated samples without prior fractionation or fractionated the samples into membrane and cytosolic fractions before they were applied.
The lead is closed to protect the fluorescent samples from light. The instrument was programmed according to recommendation and run overnight. Large 12%lamby gels were cost using adult 12 gel.
Coster displacing solution was added to avoid polymerized acry gels in the tubings. The gels were allowed to polymerize overnight at room temperature prior to use. After the isoelectric focusing, the strips are removed and equilibrating in SDS containing buffer in two steps using DTT to reduce the di sulfide bonds of cysteine residues followed by alkylation with your acetamide to avoid modification by acrylamide.
The IPG strips are dipped in running buffer and carefully placed on top of the large two dig gels. Avoid trapping air between the strips and the gel sealed by adding melted 2%agro solution with bromo anol blue on top. The gels are now ready for second dimension SDS page separation In the second dimension SDS page.
The proteins will be separated according to molecular weight and this is performed with tonal six system. Fill the electrophoresis chamber with anodic running buffer, insert the gels and fill the top compartment with cathodic running buffer program. The power supply according to recommendations and run the second dimension protected from light for approximately four to five hours or until the dye front reaches the bottom of the gel.
After the second dimension electrophoresis, the gel cassettes are placed by using the grippers in the typhoon fluorescent imager. Two gels and three channels can be scanned simultaneously. The result from this two DL shows high resolution of membrane proteins in the sample, even if there are some known restrictions for hydrophobic proteins to be detected in A two DL.The results show many new cell surface label spot shown here in red that are not detected using the standard labeling protocol shown here in green.
This results show that the cell surface labeling protocol is highly specific for labeling cell surface proteins. Since cell surface proteins are exclusively labeled, they are more easily visualized and attenuation by abundant cytosolic proteins is avoided. The fluorescent image of gels with non fractionated membrane fraction or cytosolic fraction is shown on top.
Below is an image of same gel post stain with silver. The results show no fluorescent labeling of cytosolic proteins, but the silver staining shows that there are proteins in the gels. The results also show similar spot map patterns from non fractionated and membrane fractions demonstrating that there is no need for fractionation prior to 2D electrophoresis, which makes this protocol both simple and convenient.
SI two, SI three and SCI five show similar labeling patterns and are all compatible with the cells surface protocol. A diet experiment was performed using all three side eye DI floor minimal dy for studying differential expression of cell surface proteins in CHO cells of the serum starvation for different lengths of time. CY two cell surface samples from all samples in the experiment were pooled and used as an internal standard.
The differential changes of several cell surface proteins could be followed during the starvation period. Over 18 novel membrane proteins were detected using the cell surface protocol that were not detected with the standard protocol for finding the identity of the proteins in the preparative gel. It was necessary to spike with the cell surface sample to facilitate matching back to the spots on the analytical dataset.
There results. Results show a high resolution of a large number of cell surface proteins. This protocol is very specific for cell surface proteins and no labeling of intracellular proteins were seen.
With this method, you're able to detect more cell surface protein compared to a standard protocol, and as you already seen, it's a very simple protocol to run. You simply label your cell surface proteins while they're still intact and run them directly on 2D electrophoresis without membrane fractioning. So this is a very good protocol to study cell surface proteins and the differences of those with the D technology.
