This work was supported by the National Natural Science Foundation of Jilin Province (no. YDZJ202301ZYTS189).

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Changchun University of Chinese Medicine. The materials are listed in the Table of Materials.
1. Animals and treatment
<ol>
	<li>Randomly divide a total of 50 adult spontaneously hypertensive rats (SHRs) (male, 50 days old) into five groups, including control (CON), methionine (MET), MET + HTJDTLD + Enalapril maleate (EM), MET + EM, and MET + HTJDTLD groups. 
	NOTE: The experimental rats were housed in a controlled environment designed to ensure their comfort and well-being. The facility featured temperature-controlled rooms set at a constant 22 &#176;C, with a relative humidity of 40-60%. The housing cages were made of transparent polycarbonate, providing ample space for each rat to move freely. The lighting schedule followed a 12-h&#160;light/dark cycle, simulating natural daylight conditions. The rats were provided with adequate food and water.</li>
	<li>Provide the rats with the following diet for 28 days.
	<ol>
		<li>Give the rats in the MET group a 3% methionine diet for 28 days to induce H-type hypertension.</li>
		<li>Administer the rats in the MET + HTJDTLD + EM group intragastrically with HTJDTLD (1.633 g/mL) and EM (0.2 mg/mL) at the dose of 1 mL/kg when the rats in addition to the 3% methionine diet.</li>
		<li>Administer the rats in the MET +EM and MET + HTJDTLD groups with HTJDTLD or EM by gavage, respectively, besides the 3% methionine diet. 
		NOTE: The HTJDTLD consists of Fructus Trichosanthis (20 g), Radix et Rhizoma Salviae miltiorrhizae (15 g), Lonicerae japonicae flos (30 g), Radix et Rhizoma Nardostachyos (15 g), Radix Angelicae sinensis (15 g), Radix et Rhizoma Glycyrrhizae (10 g), Hirudo (5 g), Radix et Rhizoma Rhodiolae crenulatae (15 g), and Radix Scrophulariae (15 g), and was decocted as previous reported7. EM dissolved in purified water (0.2 mg/mL) served as a positive control.</li>
	</ol>
	</li>
</ol>
2. Blood pressure measurement
NOTE: Blood pressure measurements are performed using a noninvasive sphygmomanometer (Table of Materials).
<ol>
	<li>After treatment for 28 days, fast the rats in each group for 12 h and measure the blood pressure.</li>
	<li>Choose a restraining device that matches the size of the rat. The restraining device used in this study comprises a cylindrical restraint mesh, canvas cover, thermal tube, and stabilizing foam pad. Place the rat in the restraint mesh, put the restraint mesh into the thermal tube, and then put the thermal tube into the canvas cover.</li>
	<li>Place the signal cable in a suitable position under the cover. Place the rat&#39;s tail in the gap provided in the cover and secure it on the stabilizing form pad. An unoccupied, quiet, and warm environment is preferred for measurements. Move the rats to the measurement site 20-30 min in advance so that the rats can adapt to the measurement environment. 
	NOTE: Steps 2.2-2.3 can be performed several times to stabilize the rats. After several training sessions, the rats will get used to it and can stabilize very quickly, which is convenient for the subsequent blood pressure measurement.</li>
	<li>Connect the pressurized sensor&#39;s air hose connection, signal connection, and holding tube. Place the pressurization sensor at the tip of the tail.</li>
	<li>Measure the blood pressure. A pulse wave appears after the rat tail is inserted into the sensor. Press Start/Stop to start/stop the measurement. 
	NOTE: The device will automatically determine whether the rat&#39;s tail is inserted into the sensor. When the rat&#39;s tail is not inserted, the pressurization sensor will not start pressurizing and will not make the measurement.</li>
	<li>When the blood pressure test is completed, the Result menu will pop up automatically. Check the average value of the measurement, standard deviation (SD), standard error (SE), and coefficient of variation (CV) in the Results menu. 
	NOTE: The blood pressure of each rat was measured three times, with an interval of more than 2 min, and the mean value was calculated.</li>
	<li>After measuring the blood pressure,&#160;euthanize the rat&#160;by intraperitoneal injection of excess 2% sodium pentobarbital (100 mg/kg). Then,&#160;using surgical scissors and toothed forceps, dissect the rat&#160;layer by layer from the perineum to the neck. Turn over the thoracic and abdominal contents, navigate to the abdominal aortic bifurcation between the two kidneys, and collect the aorta up to a section of the aortic arch.</li>
</ol>
3. Hematoxylin-eosin (HE) staining
<ol>
	<li>Fix the aortic vascular tissue in 4% paraformaldehyde for at least 24 h.</li>
	<li>Take the vascular specimens, dehydrate them in gradient alcohol, make them transparent in xylene, and embed them in paraffin.</li>
	<li>After embedding, make continuous slices with a 3-5 mm thickness. Dry the slices at 60-70 &#176;C, then store them at room temperature (RT).</li>
	<li>Dewax the sections by immersing the sections in xylene I for 30 min, xylene II for 30 min, 100% alcohol I for 10 min, 100% alcohol II for 10 min, 95% alcohol I for 5 min, 95% alcohol II for 5 min, 80% alcohol for 5 min, and then rinse with distilled water.</li>
	<li>Stain the sections in Harris hematoxylin for 5 min, and rinse in tap water for 5 min. Differentiate in 1% hydrochloric acid alcohol for 10 s, and rinse thoroughly in tap water for 15 min. Wash the section in ammonia water for 5 s, and rinse thoroughly in tap water for 15 min.</li>
	<li>Perform microscopic observation, stain using eosin for 10 min, and rinse thoroughly in tap water for 15 min.</li>
	<li>Dehydrate the sections by immersing them in 80% ethyl alcohol for 10 s, 95% alcohol I for 5 min, 95% alcohol II for 5 min, 100% alcohol I for 10 min, 100% alcohol II for 10 min, xylene I for 10 min, and xylene II for 10 min.</li>
	<li>Seal the sections using neutral gum.</li>
	<li>Observe the histological changes in the aorta tissues under a light microscope (100x objective, 1000x magnification).</li>
</ol>
4. Masson&#39;s trichrome staining
<ol>
	<li>Perform routine dewaxing similar to HE staining.</li>
	<li>Hematoxylin staining: Immerse the slides into hematoxylin solution for 10 min, then rinse with tap water immediately.</li>
	<li>Immerse the slides into 1% hydrochloric acid solution for a few seconds, then rinse with tap water immediately afterward.</li>
	<li>Immerse the slides into Lichtenstein acidic magenta stain for 5 min and then rinse with water.</li>
	<li>Immerse the slides into 1% phosphomolybdic acid for 5 min.</li>
	<li>Immerse the slides into 2% aniline blue staining for 5 min and 1% glacial acetic acid immersion washing solution for 1 min. Then, drop wash the slides rapidly in 95% alcohol 3 times.</li>
	<li>Perform routine dehydration, transparency, and neutral gum sealing similar to HE staining.</li>
</ol>
5. Hcy measurement by ELISA
<ol>
	<li>Anesthetize the rats by intraperitoneal injection of 2% pentobarbital sodium (45 mg/kg) and confirm the depth of anesthesia by a toe pinch.&#160;Hold the rat and cut off the whiskers to avoid contact when taking blood. Remove the eyeballs of the rat&#160;with curved forceps and collect the blood in the prepared sterile microcentrifuge tubes.&#160;And then, euthanize the rat with an intraperitoneal injection of excess 2% sodium pentobarbital (100 mg/kg).</li>
	<li>Allow the blood to naturally coagulate for 10-20 min at RT, and then centrifuge (626-1409 x g) the blood for about 20 min at 2-8 &#176;C.</li>
	<li>Carefully collect the supernatant and store it in the refrigerator at -80 &#176;C.</li>
	<li>Dilute the standard in the test tube according to the kit instructions.</li>
	<li>Set up blank wells (no samples and enzyme reagents are added to the blank control wells; the rest of the steps are the same), standard wells, and sample wells to be tested. Add 50 &#181;L of standards into the plate, 40 &#181;L of sample dilution solution into the sample wells, and then 10 &#181;L of serum (the final dilution of the sample is 5 times). 
	NOTE: Add the sample to the bottom of the wells of the plate; try not to touch the walls of the wells, and shake gently to mix.</li>
	<li>Seal the plate with sealing film and incubate at 37 &#176;C for 30 min.</li>
	<li>Dilute the 30 times concentrated washing solution 30 times with distilled water and prepare for use.</li>
	<li>Remove the sealing film carefully, discard the liquid, and shake it to remove all the liquid. Fill each well with washing solution, leave it for 30 s, and discard it. Repeat this 5 times and tap it dry.</li>
	<li>Add 50 &#181;L of enzyme reagent to each well, except the blank wells.</li>
	<li>Use a sealing film to seal the plate and then incubate at 37 &#176;C for 30 min.</li>
	<li>Repeat step 5.8.</li>
	<li>Add 50 &#181;L of color developer A to each well, then add 50 &#181;L of color developer B, and shake gently to mix well. Incubate at 37 &#176;C for 10 min under low light.</li>
	<li>Add 50 &#181;L of the stop solution to each well to terminate the reaction (the blue color will turn yellow).</li>
	<li>Zero the reaction with blank wells and measure the absorbance (OD value) of each well sequentially at 450 nm. 
	NOTE: Measurement should be carried out within 15 min after the addition of the stop solution.</li>
</ol>
6. RNA extraction and quantitative RT-PCR
<ol>
	<li>Extract total RNA.
	<ol>
		<li>Cut the fresh aortic tissue quickly to the appropriate size (30-50 mg/piece), and grind thoroughly in liquid nitrogen. Add 1 mL of Trizol reagent, mix well, and incubate on ice for 10 min to lyze the tissue.</li>
		<li>Centrifuge at 2250 x g for 10 min at 4 &#176;C. Carefully transfer the supernatant to a new microcentrifuge tube without pellet.</li>
		<li>Add 200 &#181;L of chloroform, shake vigorously for 15 s, and incubate at RT for 5 min.</li>
		<li>Centrifuge at 2250 x g for 15 min at 4 &#176;C. The mixture will be divided into three layers: the bottom phenol-chloroform organic phase, the middle phase, and the upper aqueous phase.</li>
		<li>Carefully transfer the upper aqueous phase to a new microcentrifuge tube (about 60% volume of Trizol). Do not aspirate the intermediate phase; a small amount of upper liquid can be left.</li>
		<li>Add an equal volume of isopropanol, mix gently by inverting about 10 times, and leave for 10 min at RT.</li>
		<li>Centrifuge at 2250 x g for 10 min at 4 &#176;C. Discard the supernatant and wash the RNA precipitate twice with 1 mL of 75% ethanol.</li>
		<li>Centrifuge at 2250 x g for 5 min at 4 &#176;C, discard the supernatant, and air-dry at RT for 5-10 min.</li>
		<li>Dissolve RNA in 15-50 &#181;L of diethylpyrocarbonate (DEPC) water and check the RNA concentration.</li>
	</ol>
	</li>
	<li>Perform reverse transcription and RT-qPCR (Table 1)
	<ol>
		<li>Detect the gene expression related to endoplasmic reticulum stress (ERS) and apoptosis by qRT-PCR. Use the total RNA extracted in step 6.1 and reverse transcribe it to cDNA using the cDNA synthesis kit according to the manufacturer&#39;s instructions.</li>
		<li>Determine the gene expression using a real-time PCR detection system with a SYBR Green PCR master mix in a reaction volume of 20 &#181;L.</li>
		<li>Quantitatively analyze the data by the 2&#8722;&#916;&#916;CTmethod and express it as an n-fold difference relative to the expression of &#946;-actin. The primers are shown in Table 2.</li>
	</ol>
	</li>
</ol>
7. Western blotting (WB)
<ol>
	<li>Extract total tissue protein.
	<ol>
		<li>Place a small amount of tissue block in the spherical part of 1-2 mL homogenizer, and cut the tissue block as much as possible with clean scissors.</li>
		<li>Add 400 &#181;L (w:v=1:10) of the RIPA lysis buffer and homogenize. Then, place it on ice. After a few minutes, grind again and place on ice. Repeat the grinding process several times.</li>
		<li>After 30 min of lysis, use a pipette to transfer the lysate to a 1.5 mL centrifuge tube and place the tube in a pre-cooled 4 &#176;C centrifuge. Centrifuge at 2250 x g for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.</li>
	</ol>
	</li>
	<li>Quantify the protein.
	<ol>
		<li>Dilute the protein standards according to the instructions of the kit. Obtain gradients of standards as follows: 0, 25, 125, 250, 500, 1000, 1500, and 2000 ng/&#181;L.</li>
		<li>Take 2.5 &#181;L of the protein sample and dilute it to 25 &#181;L (10 fold) using the sample diluent.</li>
		<li>Take a 96-well plate and add 20 &#181;L of standard protein sample (according to the concentration gradient) and target protein to the wells, respectively-two wells for each target protein sample.</li>
		<li>Prepare the developing solution (ready to use) by mixing liquid A and liquid B in the ratio of 50:1, and add 200 &#181;L of developing solution to each well.</li>
		<li>Place the 96-well plate with added samples in an incubator at 37 &#176;C for 30 min.</li>
		<li>Detect the absorbance at 562 nm.</li>
		<li>Calculate the concentration of protein to be measured.
		<ol>
			<li>Take the standard protein concentration as the vertical coordinate and the absorbance at 562 nm as the horizontal coordinate to draw the standard curve.</li>
			<li>Calculate the concentration of the target protein based on the formula obtained from the standard curve and the measured absorbance of the target protein.</li>
		</ol>
		</li>
		<li>Add 180 &#181;L of loading buffer to 20 &#181;L of protein sample in a microcentrifuge tube. Place the centrifuge tube in a metal bath and denature at 100 &#176;C for 10 min. Use the denatured protein WB or store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Perform immunoblotting.
	<ol>
		<li>Configure protein electrophoresis gel.
		<ol>
			<li>Clean and blow-dry the gel casting glass plate (1 mm or 1.5 mm) and fix it on the gel-casting device.</li>
			<li>Prepare 10% separation gel according to Table 3. Add the configured separating gel between the glass plates. Add the gel solution slowly to avoid the production of air bubbles. Then, add an appropriate amount of anhydrous ethanol to flatten the liquid surface of the separator. Leave it at RT for 20-30 min until the gel is solidified. 
			NOTE: The higher the molecular weight, the lower the concentration of glue, according to the need to formulate other concentrations of separating glue.</li>
			<li>Prepare 5% concentrated gel according to Table 3. After the solidification of the separation gel, pour off the upper layer of anhydrous ethanol, fill up the concentrated gel, and slowly insert the gel comb that matches the glass plate (be careful not to have air bubbles). Leave it at RT for 20-30 min for the concentrated gel to solidify.</li>
		</ol>
		</li>
		<li>Perform electrophoresis.
		<ol>
			<li>Weigh 60.5 g of Tris, 375 g of glycine, and 20 g of sodium dodecyl sulfate (SDS). Add water to 2 L, heat at 60 &#176;C, and stir to dissolve to form a 10x electrophoresis solution. Let the solution be at RT; dilute it to 1x when used.</li>
			<li>Remove the glass plate containing glue from the gel-casting device, pull down the glue comb in the water flow at a uniform speed, and at the same time, drain the air bubbles in the sampling hole.</li>
			<li>Fix the glass plate in the electrophoresis tank, and add the configured 1x electrophoresis solution. Ensure that the inner tank is full and that the outer tank is half the inner tank.</li>
			<li>Add the same mass of protein sample and 5 &#181;L of marker (used to indicate the size of protein) in the sample addition well.</li>
			<li>Run the gel at 60 V for 20 min, then at 100 V for 90 min until the loading buffer runs to the bottom.</li>
		</ol>
		</li>
		<li>Perform membrane transfer.
		<ol>
			<li>Weigh 60 g of Tris and 288 g of glycine, and add water to 2 L. Stir and dissolve the solution well to make 10x membrane transfer solution, and reserve at RT. Dilute at a ratio of 1:2:7 (10x transmembrane solution: methanol: water) to make 1x working solution. 
			NOTE: Transmembrane solution should be prepared in advance and cooled to 4 &#176;C in the refrigerator.</li>
			<li>Soak the PVDF in methanol for an appropriate period of time (0.5-1 min) to activate the positively charged groups on the membrane and make it easier to bind with negatively charged proteins.</li>
			<li>Take the membrane transfer clip and fix it after placing the components in order (black surface-sponge-filter paper-electrophoresis gel-PVDF membrane-filter paper-sponge-white surface in sequence). 
			NOTE: Be careful to avoid air bubbles; when there are air bubbles, use the roller to drive out the air bubbles.</li>
			<li>Place the splint in the membrane transfer tank, pay attention to following the correct electrode placement, put two ice bags in the tank, and fill up with 1x membrane transfer liquid. Finally, place the whole transmembrane tank in ice.</li>
			<li>Set the membrane transfer conditions to 100 V for 1 h. 
			NOTE: The membrane transfer time can be adjusted according to the size of the protein; the smaller the molecular weight of the protein, the shorter the membrane transfer time.</li>
		</ol>
		</li>
		<li>Perform blocking.
		<ol>
			<li>For phosphorylated proteins, use 5% BSA (solvent TBST) as the blocking solution. For non-phosphorylated proteins, use 5% skimmed milk (solvent TBST) for blocking.</li>
			<li>Pour the blocking solution into a dish of appropriate size. Put the PVDF membrane into the dish, and ensure that the PVDF membrane is submerged in the blocking solution. Close the dish and incubate at RT for 1 h.</li>
			<li>Remove the membrane. According to the marker display and the size of each target protein, cut the membrane into the appropriate size and label it with serial numbers.</li>
		</ol>
		</li>
		<li>Incubate the antibody
		<ol>
			<li>Remove the blocking solution and absorb the residual liquid with filter paper. Place the corresponding cut PVDF into the corresponding antibody dilutions (including CPR78 [1:1000], TRAF2 [1:1000], p-JNK [1:1000], caspase [1:1000], and GAPDH [1:1000]) and place it on the rotary shaker at 4 &#176;C overnight.</li>
			<li>Add 1x TBST and rinse at RT three times (10 min each).</li>
			<li>Incubate the PVDF membrane in the secondary antibody dilutions per the manufacturer&#39;s instructions and incubate at RT for 1 h.</li>
			<li>Rinse the membrane by adding 1x TBST at RT three times (10 min each).</li>
		</ol>
		</li>
		<li>Develop the PVDF membrane.
		<ol>
			<li>Mix equal volumes of liquid A and liquid B in the luminescent solution kit, shake well, and prepare for use.</li>
			<li>Place the PVDF membrane on the cling film to spread, and quickly and evenly add the prepared luminescent agent onto the membrane quickly and evenly. Incubate for 3 min at RT, avoiding light.</li>
			<li>Detect the protein bands using enhanced chemiluminescence (ECL) western blotting detection reagents.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
8. Data analysis
<ol>
	<li>Express all data as the mean &#177; S.D. Perform statistical analysis by one-way ANOVA using SPSS 20.0 software. Consider the differences statistically significant when p &#60; 0.05.</li>
</ol>

Antihypertensive Effects of Huotan Jiedu Tongluo in H-Type Hypertensive Rats

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+folic+acid+deficiency+is+a+major+determinant+of+hyperhomocysteinemia+in+Parkinson's+disease.">Evidence that folic acid deficiency is a major determinant of hyperhomocysteinemia in Parkinson's disease.</a> Metab Brain Dis. 24 (2), 257-269 (2009).</li><li>Zhao, W., Gao, F., Lv, L., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+hypertension+and+homocysteine+increases+the+risk+of+mortality+among+middle-aged+and+older+population+in+the+United+States.">The interaction of hypertension and homocysteine increases the risk of mortality among middle-aged and older population in the United States.</a> J Hypertens. 40 (2), 254-263 (2022).</li><li>Woo, K. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+stress+signals+in+the+tumour+and+its+microenvironment.">Endoplasmic reticulum stress signals in the tumour and its microenvironment.</a> Nat Rev Cancer. 21 (2), 71-88 (2021).</li><li>Ji, C., Kaplowitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperhomocysteinemia,+endoplasmic+reticulum+stress,+and+alcoholic+liver+injury.">Hyperhomocysteinemia, endoplasmic reticulum stress, and alcoholic liver injury.</a> World J Gastroentero. 10 (12), 1699-1708 (2004).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homocysteine+induces+programmed+cell+death+in+human+vascular+endothelial+cells+through+activation+of+the+unfolded+protein+response.">Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response.</a> J Biol Chem. 276 (38), 35867-35874 (2001).</li><li>Cunard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+stress,+a+driver+or+an+innocent+bystander+in+endothelial+dysfunction+associated+with+hypertension.">Endoplasmic reticulum stress, a driver or an innocent bystander in endothelial dysfunction associated with hypertension.</a> Cur Hypertens Rep. 19 (8), 64 (2017).</li><li>Casas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GRP78+at+the+centre+of+the+stage+in+cancer+and+neuroprotection.">GRP78 at the centre of the stage in cancer and neuroprotection.</a> Front Neurosci. 11, 177 (2017).</li><li>Urano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+stress+in+the+ER+to+activation+of+JNK+protein+kinases+by+transmembrane+protein+kinase+ire1.">Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase ire1.</a> Science. 287 (5453), 664-666 (2000).</li><li>Borghi, A., Verstrepen, L., Beyaert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAF2+multitasking+in+tnf+receptor-induced+signaling+to+nf-&#954;b,+map+kinases+and+cell+death.">TRAF2 multitasking in tnf receptor-induced signaling to nf-&#954;b, map kinases and cell death.</a> Biochem Pharmacol. 116, 1-10 (2016).</li><li>Wen, X. -. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butylphthalide+suppresses+neuronal+cells+apoptosis+and+inhibits+JNK-caspase3+signaling+pathway+after+brain+ischemia+/reperfusion+in+rats.">Butylphthalide suppresses neuronal cells apoptosis and inhibits JNK-caspase3 signaling pathway after brain ischemia /reperfusion in rats.</a> Cell Mol Neurobiol. 36 (7), 1087-1095 (2016).</li><li>Jin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine-threonine+protein+kinase+activation+may+be+an+effective+target+for+reducing+neuronal+apoptosis+after+spinal+cord+injury.">Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury.</a> Neural Regen Res. 10 (11), 1830-1835 (2015).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+propofol+combination+therapy+inhibits+endoplasmic+reticulum+stress+and+remarkably+attenuates+cerebral+ischemia-reperfusion+injury+and+ogd+injury+in+hippocampus.">Estrogen and propofol combination therapy inhibits endoplasmic reticulum stress and remarkably attenuates cerebral ischemia-reperfusion injury and ogd injury in hippocampus.</a> Biomed Pharmacother. 108, 1596-1606 (2018).</li></ol>

We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Hypertension is one of the most common cardiovascular disorders that affects one-third of the adult population and increases the risk of stroke, coronary heart disease, and heart and renal failure8. H-type hypertension is a special type of hypertension that refers to the co-occurrence of primary hypertension and increased homocysteine levels and has attracted broad attention over the years9. In recent years, the use of oral EM combined with antihypertensive drugs has shown ...

Hypertension, a major risk factor for heart attack, stroke, and renal failure, has become a significant public health challenge that affects 1 billion people worldwide1. Homocysteine (Hcy), a thiol group-containing amino acid, is a vital metabolic intermediary of methionine metabolism. Hypertension with elevated plasma Hcy levels is defined as H-type hypertension, which could be a significant risk factor for the occurrence and recurrence of cardiocerebrovascular diseases such as stroke2,3. Recent studies have reported that the co-residency of H-type hypertension could aggravate the side effects of cardiovascular and cerebrovascular diseases4. Notably, 75% of patients in China with H-type hypertension have primary hypertension, which seriously affects the quality of life5. At present, the treatment of H-type hypertension mainly includes Western medicine. However, it may cause certain adverse effects and poor compliance and can no longer meet the needs for the comprehensive management of H-type hypertension.
Traditional Chinese medicine (TCM) is a unique resource with a history of more than 2,000 years in China. Due to the unmet need for hypertension control in Western medicine, clinicians have begun to consider the potential role of TCM in the prevention and treatment of H-type hypertension6. Huotan Jiedu Tongluo Decoction (HTJDTLD) is a traditional Chinese medicine formula formulated by Professor Yue Deng, drawing from his extensive clinical expertise7. Over the course of more than 20 years of clinical application, HTJDTLD has demonstrated remarkable effectiveness in the treatment of cardiovascular and cerebrovascular diseases1. However, whether HTJDTLD has therapeutic effects in H-type hypertension has not been reported. Therefore, we aimed to explore the antihypertensive effects and specific mechanisms of HTJDTLD in rats with H-type hypertension and identify potential therapeutic drugs for the treatment of H-type hypertension.

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			<td>1st Strand cDNA Synthesis SuperMix for qPCR</td>
			<td>Yeasen,China</td>
			<td>11149ES</td>
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			<td>Anti-beta-actin antibody</td>
			<td>Bioss, China</td>
			<td>bs-0061R</td>
			<td></td>
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			<td>Anti-caspase-3 antibody</td>
			<td>Bioss, China</td>
			<td>bs-0081R</td>
			<td></td>
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			<td>Anti-GPR78 antibody</td>
			<td>Abcam, USA</td>
			<td>ab108513</td>
			<td></td>
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			<td>Anti-JNK antibody</td>
			<td>Abcam, USA</td>
			<td>ab76572</td>
			<td></td>
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			<td>Anti-p-JNK antibody</td>
			<td>Bioss, China</td>
			<td>bsm-52462R</td>
			<td></td>
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			<td>Anti-rabbit IgG antibody</td>
			<td>Bioss, China</td>
			<td>bs-0295G-HRP</td>
			<td></td>
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			<td>Anti-TRAF2 antibody</td>
			<td>Bioss, China</td>
			<td>bs-22372R</td>
			<td></td>
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			<td>Bio-Rad CFX96 Touch system&#160;</td>
			<td>Bio-Rad</td>
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			<td>real-time PCR detection system&#160;</td>
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			<td>ECL Western Blot Substrates</td>
			<td>Merck, MA, USA</td>
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			<td>Enalapril maleate folic acid tablets</td>
			<td>Yangzijiang Pharmaceutical Company, China</td>
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			<td>FastStart SYBR Green Master</td>
			<td>Sigma</td>
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			<td>Fructus Trichosanthis</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
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			<td>Hirudo</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
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			<td>intelligent noninvasive sphygmomanometer&#160;</td>
			<td>Beijing Softron Biotechnology company</td>
			<td>BP-2010A</td>
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			<td>Lonicerae Japonicae Flos</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
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			<td>Methionine</td>
			<td>Sigma, USA</td>
			<td>M9500</td>
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			<td>Radix Angelicae Sinensis</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
			<td></td>
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			<td>Radix et Rhizoma Glycyrrhizae</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
			<td></td>
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			<td>Radix et Rhizoma Nardostachyos</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
			<td></td>
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			<td>Radix et Rhizoma Rhodiolae Crenulatae</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
			<td></td>
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			<td>Radix et Rhizoma Salviae Miltiorrhizae</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
			<td></td>
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			<td>Radix Scrophulariae</td>
			<td>The First Affiliated Hospital of Changchun University of Traditional Chinese Medicine, China</td>
			<td>No catalog number</td>
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			<td>Rat Hcy ELISA Kits</td>
			<td>Shanghai Meimian Industrial Company, China</td>
			<td>MM-0293R2</td>
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			<td>RIPA buffer</td>
			<td>Shanghai Beyotime Biotechnology company</td>
			<td>P0013B</td>
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the antihypertensive effects and mechanisms of huotan jiedu tongluo decoction in rats with h-type hypertension

H-type hypertension, which is a specific form of hypertension characterized by elevated plasma homocysteine (Hcy) levels, has become a major public health challenge worldwide. This study investigated the hypotensive effects and underlying mechanisms of Huotan Jiedu Tongluo decoction (HTJDTLD), a highly effective traditional Chinese medicine formula commonly used to treat vascular stenosis. Methionine was used to induce H-type hypertension in rats, and HTJDTLD was administered intragastrically. Then, the systolic and diastolic blood pressures of the caudal artery of rats were measured by noninvasive rat caudal manometry. Histological assessment of the aorta was performed by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure Hcy levels, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting were used to determine the mRNA and protein levels of Glucose regulatory protein 78 (GRP78), Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), c-Jun N-terminal&#160;kinases (JNK), and caspase-3. The results showed that HTJDTLD significantly lowered blood pressure, alleviated histopathological lesions, and decreased Hcy levels after methionine treatment. Moreover, HTJDTLD significantly inhibited the gene and protein expression of GRP78, JNK, TRAF2, and caspase 3, which are involved mainly in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. Overall, the results indicated that HTJDTLD had effective antihypertensive effects in rats with H-type hypertension and revealed the antihypertensive mechanisms associated with inhibition of ER stress-induced apoptosis pathway activation.

As shown in Table 4 and Table 5, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly greater in the MET group than those in the CON group from 1 to 4 weeks. After HTJDTLD treatment, the SBP and DBP of the rats were significantly lower than those in the MET group. Notably, the combined utilization of HTJDTLD and EM had a stronger antihypertensive effect than HTJDTLD treatment alone.
According to HE staining and Masson&#39;s t...

Read this Scientific Article Protocol about The Antihypertensive Effects and Mechanisms of Huotan Jiedu Tongluo Decoction in Rats with H-Type Hypertension at JoVE.com

Author Spotlight: Exploring Huotan Jiedu Tongluo Decoction as an Antihypertensive Drug

Here, we present a protocol to induce H-type hypertension and evaluate the antihypertensive effects of Huotan Jiedu Tongluo decoction (HTJDTLD) administered intragastrically. In rats with H-type hypertension, HTJDTLD had effective antihypertensive effects, possibly associated with inhibition of endoplasmic reticulum (ER) stress-induced apoptosis pathway activation.

The Antihypertensive Effects and Mechanisms of Huotan Jiedu Tongluo Decoction in Rats with H-Type Hypertension

H-type hypertension, which is a specific form of hypertension characterized by elevated plasma homocysteine (Hcy) levels, has become a major public health ...

the-antihypertensive-effects-mechanisms-huotan-jiedu-tongluo

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Biochemistry

625 Views.  Changchun University of Chinese Medicine. Here, we present a protocol to induce H-type hypertension and evaluate the antihypertensive effects of Huotan Jiedu Tongluo decoction (HTJDTLD) administered intragastrically. In rats with H-type hypertension, HTJDTLD had effective antihypertensive effects, possibly associated with inhibition of endoplasmic reticulum (ER) stress-induced apoptosis pathway activation. 

Video: Author Spotlight: Exploring Huotan Jiedu Tongluo Decoction as an Antihypertensive Drug

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported from cancer patients, although relatively few have been tested and shown to cause functional changes in ErbBs. The ErbB receptors are dimerized and activated upon ligand binding, and dynamic conformational changes of the receptors are inherent for induction of downstream signaling. For two mutations shown experimentally to alter EGFR function, A702V and the &#916;746ELREA750 deletion mutation, we illustrate in the following protocol how molecular dynamics (MD) simulations can probe the (1) conformational stability of the mutant tyrosine kinase structure in comparison with wild-type EGFR; (2) structural consequences and conformational transitions and their relationship to observed functional changes; (3) effects of mutations on the strength of binding ATP as well as for binding between the kinase domains in the activated asymmetric dimer; and (4) effects of the mutations on key interactions within the EGFR binding site associated with the activated enzyme. The protocol provides a detailed step-by-step procedure as well as guidance that can be more generally useful for investigation of protein structures using MD simulations as a means to probe structural dynamics and the relationship to biological function.

The objective of this protocol is to use molecular dynamics simulations to examine the dynamic structural changes that occur due to activating mutations of the EGFR kinase protein.

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns.

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. ...