This protocol enables detection of a beta malters in human cortical tissue. First homogenized tissue is probe sonicated and clarified by low speed centrifugation. Next samples of reduced boiled in SDS buffer and loaded onto a poly acrylamide.
Gel proteins are transferred to a nitrocellulose membrane and the membrane is steam heated in phosphate buffered saline. Finally on an orbital shaker, the membrane is incubated overnight in primary antibody HRP conjugated secondary antibody, and then luminal peroxide reagent before exposure to x-ray film. Hi, I'm Rebecca Rosen from the laboratory of Larry Walker in the Department of Neuroscience at the Yerkes National Primate Research Center at Emory University.
Today we will show you how to detect Abeta multimers and cortical homogenous using SDS gel electrophoresis and immuno blotting with heat induced antigen retrieval. We use this procedure on our laboratory to study the levels and distribution of distinct Abeta multimers in unfixed cortical tissues from humans with Alzheimer's disease and age non-human primates. So let's get started.
Start with around a hundred milligrams of unfixed cortical tissue and down homogenize in around 400 microliters of ice cold buffer with approximately 30 even pestle strokes. For 20%weight per volume, the tissue weight to buffer volume ratio should be one milligram to four microliters. After homogenization transfer homogenates to a four milliliter polypropylene tube and probe sonicate three times five seconds at power five.
After sonication transfer homogenates to 1.5 milliliter micro centrifuge tubes and spin for five minutes At 3000 G, carefully remove clarified supernatants and store 50 microliter aliquots of these extracts at minus 80 degrees Celsius until use. Next, determine total protein concentration for freshly thawed aliquots of clarified homogenates using a bio acid or BCA assay according to the manufacturer's instructions clarified 20%cortical homogenates should have a protein concentration greater than five micrograms per microliter and can therefore be diluted one to 10 to be within range of the plate reader. For the BCA assay, prepare denaturing sample reactions with the same aliquots of clarified homogenates.
Use roughly 50 to 60 micrograms of total protein and adjust the volume of cortical homogenates accordingly, such that each contains the same total protein. Prepare samples to a total volume of 25 microliters for each 10 to 20%trice gel to be run. For each gel, prepare a positive control of 10 to a hundred nanograms of synthetic a beta 40 or a beta 42 peptide diluted in one XPBS to 10 microliters with 12.5 microliters of two XSDS sample buffer, and 2.5 microliters of 10 x reducing agent.
After preparing all samples, including a beta control vortex for five to 10 seconds and heat in a dry bath at 100 degrees Celsius for five minutes, then quickly spin all tubes to remove condensation in the cap. Load the samples onto a gel immersed in trice SDS running buffer in a gel box and load at least one well with 10 microliters of an undiluted molecular weight marker. Run the gel at a constant voltage of 125 volts for about 90 minutes or until the four kilo Dalton marker is about one centimeter from the bottom of the gel.
Carefully remove gels from the plastic cases and assemble the transfer sandwich according to the manufacturer's instructions. Pre-wet the blotting pads and point to micrometer nitrocellulose membranes with tris glycine. Transfer buffer and remove any bubbles from the blotting pads or the filter paper membrane sandwich.
Fill the inner chamber of the transfer apparatus with transfer buffer and fill the outer chamber with distilled water. Run the transfer for two to three hours at a constant amperage of 25 milliamps. When the transfer is complete, deconstruct the sandwich and place the gels into a dish filled with distilled water and the membranes into a dish filled with one XPBS for all protocol steps.
Make sure the gels and membranes are completely covered with fluid to avoid drying out. To visualize the efficiency of protein transfer stain gels with undiluted, simply blue safe stain for around one hour incubation time and membranes with one x PON S red stain for around five minutes. Incubation time according to the manufacturer's instructions, this staining will not interfere with immuno blotting.
If staining reveals inefficient protein transfer, modify the transfer time in step three for antigen retrieval. In a steamer heat seal the membrane in a heavy duty CapEx plastic pouch filled with around 50 milliliters of one XPBS at room temperature. Lay the pouch flat in a preheated steamer at 100 degrees Celsius once the pouch begins to expand, incubate for an additional 15 minutes before removing from the steamer.
Allow the membrane to cool slowly before removing it from the pouch to prevent excessive wrinkling at room temperature. Carefully remove the membrane from the pouch and rinse the membrane for five minutes and one XPBS on an orbital shaker followed by a five minute rinse in TBST on an orbital shaker. Next, incubate the membrane in blocking solution for one hour on an orbital shaker without rinsing, transfer the membrane to a dish containing the primary antibody six E 10, which is specific to the A beta and terminal region diluted one to 1000 to one to 5, 000 in blocking solution.
Incubate on an orbital shaker at room temperature for one hour and then 24 to 48 hours with shaking at four degrees Celsius. Following one quick rinse, wash the membrane for 30 minutes. In TBST follow with three 10 minute rinses on an orbital shaker.
Incubate the membrane in HRP conjugated secondary antibody diluted at one to 10, 000 in blocking solution for 90 minutes on the shaker at room temperature. In this protocol we use sheep anti-US antibody. Again, rinse the membrane for 30 minutes In TBST starting with one quick rinse and following with three 10 minute rinses on an orbital shaker.
After the last rinse, incubate the membrane in freshly made undiluted super signal West Pico ECL reagents for five minutes. On the shaker at high RPM, remove the membrane and bloss off excess reagent. Using lint-free filter paper or kin wipes, place the membrane in a film cassette between plastic sheet protectors, blot off additional super signal reagent with dust-free Kim wipes.
If necessary, expose to Kodak Biomax MR.Film for intervals of 30 seconds, up to 30 minutes and develop in a film developer. After five minutes, the A beta monomer bands will probably be saturated In this experiment. Clarified homogenates containing 50 micrograms of total protein from seven human subjects were analyzed for the presence of multimeric beta amyloid or a beta and amyloid precursor protein or a PP, the transmembrane protein from which the A beta peptide is cleaved.
Here we show representative images from an A beta a PP western blot experiment at 30 minutes and five minutes exposure. A 30 minute exposure allows for visualizing the much lighter dimer, trier, tetramer and higher molecular weight or ligament bands of Abeta, but over exposes the other lanes at five minutes exposure time. The same tetramer band is present but faint and allows other lanes to be clearly visualized.
Cortical homogenates from subjects with Alzheimer's disease or AD are seen in lanes one to three, a human subject diagnosed with dementia with livey bodies or DLB and Alzheimer's disease is seen in lane four cases of dementia with Lewy bodies are seen in lane five and six and an aged non demented human control case. In lane seven, immuno blossoming with antibody six C 10 revealed a beta monomers, dimers, trimmers, tetramers, and a PP in all Alzheimer's cases as well as abundant higher molecular weight. A beta malters in two ad cases, which is indicative of the heterogeneity of a beta ization between human subjects synthetic.
A beta 40 in the right lane confirms the identity of the lower molecular weight bands. We've just shown you an SDS page Western body experiment to identify a beta multimers in unfixed tissue homogenates from the brains of humans with neurodegenerative diseases. When performing this procedure, it's important to prepare highly concentrated cortical homogenous for separation by gel electrophoresis in order to reveal lower abundance Abeta malters.
It's also important to perform heat induced antigen epitope retrieval prior to immuno blotting with an antibody to the Abeta peptide. Antigen retrieval helps to reveal obscured Abeta epitopes for optimal antibody binding during immuno blotting. Any steamer, microwave or water bath that maintains 100 degrees Celsius will suffice.
So that's it. Thanks for watching and good luck with your experiments.
