Figure 1. In the presence of transfer buffer, place two 2 pieces of Whatman paper A soaked in transfer buffer followed by the gel with separated proteins B, the PVDF membrane C soaked in methanol then transfer buffer, and two 2 more pieces of soaked Whatman papers. Arrow shows the direction of protein transfer. An oversized piece of PVDF membrane, to cover the entire gel, is soaked in methanol first followed by transfer buffer and then placed on top of the gel. Sandwich is disassembled, membrane is removed and any extra piece of membrane that is bigger than the gel is cut out.
Wash membrane 3x by adding 20 ml of washing solution and slow rocking for 5 min to remove excess BSA. Then, incubate the membrane with the primary solution biotinylated lectin in 0. Wash membrane 3x followed by the addition of the secondary solution HRP-streptavidin in 0. After incubation with secondary solution, wash the membrane 4x to visualize biotinylated lectins bound to glycoproteins using enhanced chemiluminescent detection kit, mix equal volumes of detection solution 1 and detection solution 2 for a final volume of 0. Drain excess detection solution and place membrane in an autoradiography cassette for film exposure.
Due to the variability of carbohydrate content on glycoproteins, different exposure of the membrane to the film should be performed. Representative data A representative image of Cnm analysis for different strains of S. Notes Due to the variability of the type and the level of glycosylation for each protein, protocol optimization may be required for successful identification of glycoproteins by lectin analysis. Control glycoproteins with known reactivity to specific lectins should be included in each experiment. Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence.
Lectin Methods and Protocols (Methods in Molecular Medicine) - PDF Free Download
J Bacteriol 15 : Nothaft, H. Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8 11 : Bio-protocol 5 7 : e Electron Microscopy. Madrid, Francisco Hernandez, and Jose Ballesta. Mitchell and Udo Schumacher. Part II. Gram and John-Erik Stig Hansen. Part III. Part IV. Use of Lectins in Quantification of Soluble Glycoproteins.
Quantification of Intestinal Mucins, Jeremy D. Milton and Jonathan M. Part V. Lectins in Affinity Purification of Soluble Glycoproteins. Kerr, Lesley M. Loomes, Brian C. The alternative printing protocol could take either more or less time depending on the number of tips used and the print design. For Basic Protocol 2, if the samples are already prepared, the hybridization and scanning will take approximately 5 hours. Sample preparation times for glycoproteins and cell membranes vary. We suggest overnight dialysis for cell membrane samples. Including the ultracentrifugation, labeling and dialysis, cell membrane sample preparation should take approximately 15—20 hours.
This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article. Curr Protoc Chem Biol. Author manuscript; available in PMC Feb 1. PMID: Kanoelani T. Mahal 1. Lara K.
Author for correspondence: Lara K. Copyright notice. The publisher's final edited version of this article is available at Curr Protoc Chem Biol. See other articles in PMC that cite the published article. Abstract Lectin microarray technology has been used to profile the glycosylation of a multitude of biological and clinical samples, leading to new clinical biomarkers and advances in glycobiology. Keywords: carbohydrate analysis, glycomics, lectin microarray, Nano-Plotter, piezoelectric. Open in a separate window. Figure 1.
Table 1 Panel of lectins included in our lectin microarray organized by carbohydrate specificity. Lectin source Abbreviation Print conc. Advantages of non-contact printing In contrast to genomics based microarrays where the most widely used platform is produced by an in situ synthesis technique Hardiman, , protein arrays are typically manufactured using either a contact or non-contact printing process that transfers samples from microwell plates to a solid support, typically a derivatized glass slide.
Experimental design: single versus dual color analysis Typically, we hybridize samples for both single and dual color analysis. Equipment Nano-Plotter 2. Figure 2. The slides must be allowed to thaw to room temperature to avoid condensation on the slide surface, which can result in loss of activity. The addition of a complementary monosaccharide helps to prevent aggregation during the print process. We have found it best to make a fresh plate for each print. Reuse of plates is not advised as most lectins lose activity when stored in the fridge see Critical Parameters for more on this subject.
Determine the number of slides to print. Take out the slides from deep freezer and keep at room temperature for 30 minutes prior to printing. Set-up of Nano-Plotter: Check the humidity of the print chamber. A humidity control device may be used to maintain the appropriate relative humidity see Strategic Planning section. This is important to help maintain lectin activity during extended and overnight print runs. Once the temperature and humidity reach the desired parameters, start the print setup See troubleshooting point 1.
Refer to the Nano-Plotter manual section 2. Turn on the machine and accessories Nano-Plotter and the Diluter, Figure 2. For the Nano-Plotter, you should hear two beeps See troubleshooting point 2. In our prints, we have found that printing with 2 tips is prone to artifacts, including tip-to-tip variations in deposition and positional defects, thus we do not recommend printing with multiple tips.
Instead, for the best quality arrays, we print with a single tip using the more advanced Multitask Program. Sequential program allows you to wash and aspirate two tips at the same time Refer to the Nano-Plotter manual section 7. During initialization the Nano-Plotter goes through a system check.
Lectin Methods and Protocols
If successful, the NPC print program can be accessed. In interactive mode, all built-in functions and devices of the Nano-Plotter can be activated in a direct interactive manner. This is useful if you want to perform adjustments, washing cycles and a pipette test before starting a program. Run mode is the mode you must be in to run a print program. Simulate mode, allows you to simulate the program generated visually in NPC.
These menus are easily accessible from either the pull down menus or the button bar at the top of the program window refer to the Nano-Plotter manual section 3. Activate and assign tips, tip sockets and diluter positions. Select the tip IDs or enter a new one by pressing the button to the right of the field. Select the sockets and the diluters you are using. The socket is the electrical socket that the tip is connected to. The diluter is the fluidics system, where 1 is the tube closest to the front of the machine. Make sure that you have the tip you just selected assigned to the proper location by clicking on that position in the Mechanical Assignment box.
This should correspond to the physical tip assignment in the print head. Position A is closest to the front of the machine. Connect the tubes to the tips. Make sure the tips are dispensing correctly. For the Nano-Plotter, use the Stroboscope to check how the tips are dispensing. If they are dispensing without sputtering, they are probably fine See troubleshooting point 5. At this point, the voltage can be set for water, although it will be adjusted for lectins later. The ideal voltage may vary with tip but the stroboscope pattern should be two to three drops in a line with some space in between them Figure 3.
Figure 3. Generate the print design parameters: For the Nano-Plotter, select the workplate. Any type of target can be accommodated in the workplate. Right clicking on the targets in Edit mode will give you a menu to edit or add new targets or the spot layout. The module for slides as a target group already exists, thus the dimensions are stored and you can increase the number of slides easily by increasing the rows and columns of slides. Edit the target number to reflect the number of slides you would like to print. Edit the spot layout in order to use the Sequential.
If a target or target group contains more than one block then make appropriate entries such as rows of blocks, columns of blocks, distances between block row and block column. In Sequential, the number of spot columns and rows must be a multiple of the lectin columns and rows in the microwell plate.
We normally print 3 replicate spots of each lectin. Thus for a row with 5 lectins, the spot layout should be 15 columns. We leave enough room on either side of the array to aspirate buffers without scratching the printed arrays during the washing steps after hybridization, typically 1. Load the appropriate number of slides onto the printer deck in the orientation defined in the Workplate file. Make sure the Z height is set for the print substrate. In the Nano-Plotter, use a Z sensor test. A Z-Level-Sensor, which can be attached to the side of the printhead, gives the exact height of each object on the tray.
Although piezoelectric dispensing is always non-contact, close proximity between slides and dispenser nozzle ensures well-aligned spots. A popup window will ask you to mark the microwell plate to indicate the number of rows and columns in the microplate that are filled with lectins.
The two tips then dispense sequentially in a pattern that is a direct copy of the loaded samples in the microwell plate. See troubleshootin g point 6. We typically fill 5—6 columns and all 16 rows of the microplate in order to accommodate 80—90 lectins.
The number of replicates for each sample is determined from the number of spots designated for the target slides in the workplate Figure 4 Refer to the Nano-Plotter manual Section 7. If you assign 5 wells of sample to the microplate and 15 spots to the slides, Sequential will assume that you want 3 spots of each lectin.
Therefore, the number of spots must be a multiple of the number of wells or you will receive an error. A pop-up window will confirm that you want 2 replicates in addition to the single default spot per sample. Figure 4. In the next popup window, you will have the option to change several variables including number of droplets of the replicates, stroboscope delay time, distance to target, extra sample volume, aspiration flow, wash time and stroboscope break Table 2.
Refer to the Nano-Plotter manual Section 7. We use a wash time of 30 seconds and recommend wash times between 30—60 seconds for the best results. We recommend performing the StroboCheck, as lectins are often more viscous than water and voltage adjustments may be required. Lectins that do not pass the StroboCheck will be dispensed to waste and their microwell plate coordinates will be saved to the RepairSeq. Check the distance of the pipettes to your targets.
A value of 0. Considerably higher values may be used, however this can lead to scattering due to static electricity. Lower values can also be used but a distance of at least 0. We change the height so that the pin prints at 0. This may not be the case for all slides or surfaces and it is recommended that you test them with the dummy tip first. The extra sample volume prevents the sample from being diluted with system liquid there is no air gap between sample and system fluid. An extra sample volume between 0. With a larger volume, the percentage change in concentration due to evaporation is lower.
Stroboscope break: The stroboscope break is quite important to adjust the piezo parameters of the pipettes for the real samples, which will generally be more viscous than water. Change the parameters such as voltage and frequency until optimal drops are produced Figure 3. We perform the Stroboscope break to adjust the parameters for the first lectin. Although lectin properties can vary and influence the drop pattern, it is complex to change the parameters for each lectin.
However, it is possible to perform a Stroboscope break periodically as the print is running. If you know that a particular sample is more viscous, you can readjust the parameters using the Stroboscope break. The program should commence. Samples that do not pass the Stroboscope check are not printed and the program marks the graphic representation of the microplate with an orange circle to indicate the missing sample. Periodically monitor the printing process to make sure that everything is functioning properly.
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After the print is finished, we wash the tips several times and check the stroboscope until we get a good droplet distribution without stray spots fluttering before running the correction file. A correction file is written during the print with the microwell plate coordinates for the samples that did not pass the stroboscope test.
Run RepairSeq. Incubate the arrayed slides for 1 hour at room temperature post-printing, in order to maximize the protein immobilization.
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Shut down the Nano-Plotter: Wash the tips until they dispense spots without fluttering in the Stroboscope. Run the Goodnight. We find that performing this shut-down procedure minimizes clogs and air bubbles that make the next print set-up more difficult. Materials Reagents Glycoproteins Sigma, St. Louis, MO or cells harvested from tissue culture Lectin array from Basic protocol 1.
Instruments VialTweeter sonicator Heischler U. Incubate the glycoprotein for 30—45 minutes at room temperature on a rocker. Quench the free dye with mM Tris buffer pH 6. If necessary, dialyze the samples against PBS. We found dialysis helpful in reducing background with complex samples such as cell membranes, possibly due to improved micelle formation, as hybridized free dye does not result in increased background. Minimize the amount of light exposure by covering or performing in the dark.
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All steps performed after this point should take light exposure into consideration. Alternatively, isolate the membrane components of cells harvested with a cell scraper. We avoid the use of proteases, such as trypsin, to release cells to prevent any bias in the glycoprotein pool due to selective cleavage Takekawa et. Physically disrupt cells by probe tip sonication to form micellae or by sonication using a VialTweeter Heischler , which can simultaneously sonicate multiple eppendorf tubes at high cavitation allowing smaller volumes to be processed.
We have adapted our original protocol for cell membrane preparation to the VialTweeter. The resistance increases and power decreases along the length of the VialTweeter. For the best results, load two eppendorf tubes into the VialTweeter holes closest to the power source. Repeat this twice for a total of 3 times.
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We have observed that this results in the formation of membrane vesicles that are similar to those observed for probe-tip sonication. Follow by NHS-Cy dye labeling of samples as described above for glycoproteins. The labeled cell membrane preparations can be aliquoted and snap frozen in liquid N 2. Slide processing and microarray hybridization 3. Prior to use for a large experiment, one of the slides should be tested using one or many glycoprotein standards. This adds an important level of quality control that can help identify inactive and misprinted lectins.
We do this by evaluating the activity of both the whole slide and individual lectins for every block see Additional Considerations section: Glycoprotein signatures as a quality control measure. Lectin array slides should be handled very carefully. Always wear gloves during slide handling and avoid any contact with the slide surface.