Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

The overall goal of this 3D-PAGE procedure is to separate protein and ribonucleoprotein complexes from Brassica napus ploem sap, coupled with the identification of the respective nucleic acid and protein components. This method can help to answer key questions in biochemistry and plant physiology. For example, you can analyze the composition of phloem complexes under stress.

The main advantage of the technique is that pure phloem sap can be collected and nucleic acids, as well as protein components can be analyzed simultaneously. Though this method can provide insights into protein protein, and protein nucleic acid interactions within the phloem system of oilseed rape, it can also be applied to phloem analysis of other plants like, cucurbits, lupines, and yucca. We first had the idea for this method when we tried to identify protein and RNA interaction partners for specific phloem protein.

First, puncture the inflorescent stem of an eight week old B.napus plant several times using a 0.8 millimeter hypodermic needle. Puncture several other inflorescent stems on the same plant and other plants to obtain enough phloem sap. Wipe off the first drop from each injured site with filter paper.

After removing the first drops, use a pipette to collect the remaining drops. And transfer them to a pre-chilled conical 1.5 milliliter reaction tube. Then store the collected exudate at minus 20 degrees Celsius using an ice-free cooling system.

Continue collecting drops until no further drop formation is observed, but no longer than one hour. Next, add the phloem sample to a centrifugal concentrator with a molecular weight cut off of 10, 000 daltons, and concentrate to approximately 1/6 of the initial volume. Then, centrifuge the concentrated sample at four degrees Celsius and 20, 000 g for at least 15 minutes to remove dust particles.

To perform blue native PAGE, assemble the gel chamber. And add dark blue cathode buffer B to the half, and wash the wells with the cathode buffer. Load 20 to 30 micrograms of the concentrated protein sample per well on to a commercial Bis-Tris radiant gel in at least triplicate.

Fill the chamber with cathode and anode buffer, and run the gel at four degrees Celsius and 150 volts until the sample passes 1/3 of the gel, which takes 20 to 30 minutes. Pause the run an exchange dark blue cathode buffer B with light blue cathode buffer B 1/10. Restart the run and continue until the blue front starts to elute out of the gel, which takes 120 to 180 minutes.

Cut out a whole gel lane from the native blue gel, then transfer onto a nylon membrane by semi-dry blotting, and mark the upper and lower gel borders on the membrane with a pencil. After blotting, wash the membrane with DEPC-treated water and place between two pieces of filter paper to dry. Following this, perform UV cross linking of the blotted membrane with an applied energy of 120, 000 micro joules per centimeter squared.

Now, pre-hybridize the UV cross-linked membrane with six milliliters of hybridization buffer for 60 minutes at 68 degrees Celsius in a hybridization oven. Add an additional one milliliter of hybridization buffer containing four microliters of 3-prime biotinylated probes to the membrane. Then, incubate the membrane with the probe overnight while cooling the hybridization oven from 68 to 37 degrees Celsius.

On the next day, wash the membrane with buffer containing 2x SSC and 0.1%SDS for five minutes at room temperature to remove probes bound unspecifically. Perform the detection of the 3-prime biotinylated probes on the membrane with a streptavidin HRP conjugate and luminol as a horseradish peroxidase substrate, resulting in a sensitive chemiluminescent reaction. Excise single bands from the blue native gel.

Combine bands from samples run in parallel lanes in a 1.5 milliliter conical reaction tube to achieve a further concentration of complex proteins. To prepare a 12%Tris-Tricine urea gel, pour 10 milliliters of gel solution A between two glass plates and overlay with isopropanol. After polymerization, remove the isopropanol, add two to three milliliters of gel solution B to the gel, and insert a 10 well comb.

For equilibration of the excised gel pieces, add one milliliter of 2x SDS sample buffer into the reaction tube. After incubating the sample for 10 minutes at room temperature, heat in a heating block at 95 degrees Celsius for approximately one minute. Then incubate the sample at room temperature for 15 minutes.

Following this, stack several equilibrated gel pieces representing the same complex into one single gel pocket. After assembling the gel chamber, perform electrophoresis using anode and cathode running buffers at 150 volts for 45 to 60 minutes. When finished, excise the whole gel lane.

Incubate the cut gel lane for 20 minutes in acidic buffer. Then, equilibrate in 125 millimolar Tris-HCL at pH 6.8 for another 20 minutes. Pour a 15%SDS separating gel solution according to lanely.

Once the gel solidifies, remove the isopropanol, add three milliliters of a 4%stacking gel solution, and insert the special comb for IPG gels. After solidification, transfer the equilibrate gel lane onto the SDS gel, and cover the gel with 0.5%agarose in 1x SDS running buffer supplemented with a trace of bromophenol blue. After assembling the gel chamber, run the gel at 150 volts for 60 minutes.

When finished, stain the gel with colloidal Kumasi solution for subsequent mass spectrometric analysis. Finally, analyze the visible protein spots using matrix-assisted laser desorption ionization time-of-flight spectrometry. A representative result obtained from a pure phloem sample is depicted here.

Non-contaminated phloem samples show a visible band for thioredoxin age, whereas no signal for RuBisCO and the pollen coat protein is observed. At least four major protein complex bands become visible after BN PAGE of B.napus phloem sap. Too low or too high concentrations of phloem sap do not allow a clear separation of the complexes.

High resolution separation is observed in 3D PAGE due to the different protein migration behaviors in the urea and SDS-PAGE gels. Typically, proteins belonging to a single complex will be visible as a diagonal line across the gel. Proteins above this line have an increased hydrophobicity, whereas proteins below the line are more hydrophilic.

BN northern blotting shows that a specific tRNA is only present in one specific complex. Mass spectrometric analysis showed that this complex contains mainly tRNA ligases that confirms the tRNA binding activity found by the BN northern blotting. Once mastered, this technique can be done in three days if it is performed properly.

While attempting this procedure, it is important to remember to wear gloves and a lab coat to minimize carotene contamination that will hamper mass spectrometric analysis. Following this procedure, other methods like zymograms and enzyme assays with collected phloem sap can be performed in order to answer additional questions, like complex activity and inhibition studies. After its development, this technique paved the way for researchers in the field of plant science to explore multi-subunit complexes in phloem samples from different plant species.

After watching this video, you should have a good understanding of how to isolate phloem sap from Brassica napus plants, and how to isolate and analyze protein protein and protein nucleic acid complexes. Don't forget that working with SDS, acrylamide, and TMET can be extremely hazardous, and precautions such as working under hood with gloves and a lab coat should always be taken while performing this procedure.