The overall goal of this procedure is to describe a useful tool for visualization, characterization, and quantification of huntingtin protein flux for the analysis of protein homeostasis in Huntington's disease model systems. This method can help us understand questions in protein homeostasis and neurodegenerative disease fields such as understanding the impact of certain therapeutic interventions on disease pathogenesis. The main advantage of this technique is that it allows investigators to isolate and resolve insoluble protein species for quantitative analysis.
Though this method can provide insight into biochemical changes during Huntington's disease pathogenesis, it can also be applied to other protein misfolding diseases such as Parkinson's and Alzheimer's disease. To begin this procedure, label two sets of tubes for each sample as soluble and insoluble. Prepare a lysis buffer immediately prior to sample lysis in the soluble tube by adding the appropriate amount of inhibitors.
Next, add ice cold working soluble lysis buffer supplemented with protease inhibitors to the tissue. For mouse brain tissue, slowly dounce 30 times in a one milliliter glass douncer. Be careful not to break the surface of liquid as bubbles will form and may yield incomplete homogenization.
Then pipette it into the insoluble-labeled tube on ice. For cell lines, cells were plated at five times 10 to the fifth cells in each six-well plate and grown to near confluency. Afterward, rinse the cells once with cold 1X PBS and remove it.
Then apply minimal volume of lysis buffer to the cells and lift them with a cell scraper. Next, transfer homogenate or cell lysate into the labeled insoluble tube and lyse on ice for one hour. For cell lysates, triturate several times to break up cell clumps prior to starting incubation and avoid forming bubbles.
Briefly pulse vortex each sample for one second halfway through lysing. After that, centrifuge the samples at four degrees Celsius at 15, 000 x g for 20 minutes. Then remove the supernatant.
Be careful not to disturb the pellet or cloudy layer as this will contaminate the fraction. Pipette the supernatant as soluble fraction into the soluble-labeled tube and keep it on ice. Keeping track of the soluble and insoluble lysis buffers is key.
Switching the buffers or placing the insoluble buffer or samples on ice will be detrimental to the outcome of the protocol. Wash the pellet with 500 microliters of lysis buffer and centrifuge at four degrees Celsius at 15, 000 x g for five minutes. Repeat the wash one more time.
Subsequently, remove all the remaining wash buffer leaving only the tissue pellet. Next, resuspend the pellet with lysis buffer supplemented with 4%SDS. Sonicate each sample for 30 seconds at room temperature with a probe sonicator.
Sonicating small volumes for a long period of time is technically challenging. Be careful not to lose any sample by emulsifying or spraying while completing this step. Afterward, boil the samples for 30 minutes.
Then centrifuge them briefly at 6, 000 x g. Subsequently, perform detergent compatible protein assay or Lowry protein assay to measure protein concentration. Following that, aliquot the samples into 50 microliter batches to avoid freeze/thaw issues.
In this step, perform western blots by diluting 30 micrograms of sample at a ratio of one to one in loading buffer. Boil the samples for five minutes and centrifuge them briefly at 6, 000 x g. Next, analyze the fractions by SDS page and western blot.
Stain the membrane with whole protein stain to visualize protein loading efficiency. Here are the soluble and insoluble fractions from HEK 293T cell lysates resolved on a 4-12%Bis-Tris and 3-8%Tris-Acetate page gels respectively. Soluble fraction normalized to GAPDH loading control shows fluctuation of mutant huntingtin exon 1 soluble monomer based on exposure to protease inhibitor MG132.
Insoluble fraction shows that high molecular weight accumulated species of mutant huntingtin is also modulated by MG132 treatment. And here are the samples of soluble and insoluble fractions from HeLA cell lysates using western blot and filter retardation analysis. Data shows that the presence of an over expressed SUMO isoform increases both high molecular weight mutant and fibrillary insoluble mutant huntingtin in the insoluble fraction.
This figure shows the regulation of high molecular weight mutant huntingtin accumulation after modulating a potential therapeutic in the insoluble fractionation from HeLa cells over expressing 97Q huntingtin exon 1. Therapeutic target PIAS1 was either silenced with an siRNA towards PIAS1 or over expressed. Insoluble fractions from mouse striata treated with microRNA against PIAS1 and resolved on 3-8%Tris-Acetate page gels show modulation of high molecular weight insoluble huntingtin.
Don't forget that working with NEM can be extremely hazardous and taking precautions such as wearing a face mask should always be taken while performing this protocol. Once mastered, this technique from lysate to end of fractionation can be done in about three to four hours depending on the number of samples. An additional hour should be allowed for protein quantification and aliquoting.
Following this procedure, other methods such as agarose gel electrophoresis or filter retardation can be performed in order to detect soluble oligomers or insoluble fibrils respectively. After watching this video, you should have a good understanding of how to isolate and resolve insoluble misfolded proteins for quantitative analysis.
