The overall goal of the following experiment is to illustrate the use of glycosate for the enzymatic deglycosylation and analysis of n and O Linked glycosylation on a model glycoprotein. This is achieved by treating a glycoprotein with PGA's F or with the protein deglycosylation mix. Also, the protein deglycosylation mix was supplemented with a mixture of additional exo glyco ass, which sometimes help remove otherwise resistant sugars.
We illustrate this method using a model glycoprotein recombinant human chorionic gonadotropin beta, or HCG beta next SDS page, followed by Kumasi blue staining and a sugar specific staining method. Pro Q Emerald 300 are used in order to analyze the D glycosylated. HCG beta sample results are obtained, which show that HCG beta is heterogeneously glycosylated and contains multiple glyco forms based on the differences in protein migration with and without glycosate treatment and the diminished signal on ProQ staining as the glycans are successively removed.
The main advantage of this technique over existing methods, such as mass spectrometry analysis, is that it's simple, uses common laboratory equipment and is good for the novice glyco biologist. This method can answer key questions in the glyco field, such as is my protein glycosylated. To begin enzymatic deglycosylation thaw the supplied buffers from the protein deglycosylation kit.
Mix the tubes thoroughly and keep it room temperature. Place the enzyme containing vials on ice, dissolve the contents of the HCG beta vial in 600 microliters of distilled water and keep on ice. After preparing one milliliter of one XG seven buffer by diluting the 10 x stock in distilled water, use 25 microliters to dilute 0.5 microliters of PGA's f.
Prepare the exo glycosate mix by combining two microliters, each of the four exo glyco diseases. Next, add HCG beta and 10 x glycoprotein denaturing buffer to seven numbered PCR tubes as indicated in the written protocol, cap the tubes and mix gently denature the proteins in the thermocycler by incubating 10 minutes at 94 degrees Celsius, followed by a four degrees Celsius Hold After removing the tubes from the thermocycler centrifuge to remove any visible condensation to each reaction tube, add the remaining reagents according to the written protocol. Close the PCR tubes using new caps.
Then mix the tubes by gently tapping four times after spinning down the contents, place the tubes in the thermocycler incubate at 37 degrees Celsius for four hours. Follow by cooling the samples to four degrees Celsius. To set up SDS page, prepare fresh three x reducing SDS loading buffer.
With DTT, add 12.5 microliters of the prepared three x reducing SDS loading buffer To each sample, close the tubes with new caps and gently tap the tubes to mix. Incubate the tubes in a thermocycler at 94 degrees Celsius for five minutes, and then cool to four degrees Celsius Following incubation load, 30 microliters of each sample and 10 microliters of the protein marker On a 10 to 20%tri glycine gel. Saving the remainder of each sample.
Electros the gel at 130 volts until the D front is near the bottom of the gel. When the gel has finished running, remove the gel from the cast and place it in a small plastic box with enough kumasi blue stain to cover the gel stain. The gel for one hour with gentle agitation After the gel is stained.
Wash three times for 30 minutes. In 50 milliliters of detain solution, record the images using a white light transluminator or scanner. Alternatively, the gel can be dried between sheets of cellophane.
In a frame, run the remainder from each sample and a protein marker on a 10 to 20%Tri glycine gel while the gel is running. Dissolve the ProQ emerald reagent with DMF and prepare the stock fix, wash and oxidizing solutions for the stain following the product manual provided with the kit. When the electrophoresis is complete, remove the gel from the cast and place it in a plastic box.
Fix the gel by adding 100 milliliters of the prepared fixed solution. Leave the gel overnight at room temperature with gentle agitation the following day. Wash the gel with 100 milliliters of the prepared wash solution for 10 to 20 minutes at room temperature with gentle agitation, repeating the wash a second time with fresh wash solution.
Next, oxidize the carbohydrates by incubating the gel with gentle agitation for 30 minutes in 25 milliliters of the prepared oxidizing solution, follow the incubation with two additional washes while the gel is washing. Prepare fresh ProQ emerald 300 stain by adding 500 microliters of the ProQ Emerald 300 reagent solution to 25 milliliters of staining buffer provided in the kit. Stain the gel by incubating in the prepared stain under dark conditions with gentle agitation for 90 to 120 minutes subsequent to gel staining.
Repeat the two wash steps as described earlier. Then record the images with a UV trans illuminator at 300 nanometers. As a final step, compare the images of the kumasi stained gel with the ProQ emerald stain gel.
The changes in protein migration after enzymatic deglycosylation can be seen when the control sample is compared with the PGA's F treatment. To remove n glycans and to the Deglycosylation mix treatment to remove n and O glycans, no further reduction in size is seen after digesting with additional glycosate. Besides a change in mass, bands become sharper as glycans are removed.
A band running under the 17 kilodalton marker probably represents the fully deglycosylate HCG beta polypeptide. Lanes five to seven show the bands corresponding to the Glyco. Other bands might derive from incomplete deglycosylation or from multiple unidentified proteins present in the HCG beta sample.
The emerald green reagent oxidizes and stains all glycans present in a protein molecule. Therefore, the intensity of the signal decreases as HCG beta is enzymatically d glycosylated. The residual signal in lane three and four indicate the presence of glycan motifs, which are resistant to the enzymes used.
The additional glycosate is used in lane four. Remove a few extra sugar residues. The protein migration is the same, but a slight reduction in intensity in staining can be seen.
Resistant sugar moieties were not present in all protein species. Some bands were not detected by emerald Green indicating that they were extensively de glycosylated Additional data support the conclusion that HCG beta is heterogeneously glycosylated. The lower band on lane two is faint on the emerald green image.
While the upper band on lane two is bright indicating that many GLYCAN groups are still present, these data support the conclusion that recombinant HCG beta expressed in mouse cells contains multiple glyco forms due to the inherent heterogeneity of glycosylation. Following this procedure, other methods such as mass spectrometry can be performed in order to answer additional questions such as, what is the rate of occupancy? What is the extent of glycosylation, or what is the fine structure of the glycans?
After watching this video, you should have a good understanding of how to use glycosylate for the enzymatic deglycosylation and analysis of NNO Linked Glycans. On glycoproteins. The choice of detection can be difficult because protein staining regions are useful only if the de glycosylation results in a significant shift in molecular mass for other proteins.
We have seen abnormal migrations after de glycosylation and have found that any change in migration is evidence that the protein has been de glycosylated.
