Molecular weight standard

Manufactured by Bio-Rad

Sourced in United States, Spain

Molecular weight standards are laboratory tools used to determine the molecular weights of proteins or other macromolecules during electrophoresis. They provide a reference to estimate the size of unknown samples by comparing their migration patterns to the known molecular weights of the standard markers.

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Lab products found in correlation

Mini protean tgx precast gel, by Bio-Rad (2 mentions) Ecl chemiluminescence detection kit, by Cytiva (2 mentions) Iblottm dry blotting system, by Thermo Fisher Scientific (2 mentions) Mini protean system, by Bio-Rad (2 mentions) Genetools software, by Syngene (2 mentions) Image lab software, by Bio-Rad (2 mentions) 1100 hplc, by Agilent Technologies (1 mentions) Superdex 200 increase 5 150 gl column, by Cytiva (1 mentions) Superdex 200 column, by Cytiva (1 mentions) Tskgel g3000swxl, by Tosoh (1 mentions) Yarra 3 sec 3000 column, by Phenomenex (1 mentions) Goat anti human igg horseradish peroxidase, by Southern Biotech (1 mentions) Cyclone storage phosphor system, by PerkinElmer (1 mentions) Multisensitive medium ms, by PerkinElmer (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Sephacryl s 400, by GE Healthcare (1 mentions) Kta pure chromatography system, by GE Healthcare (1 mentions) Ab23875, by Abcam (1 mentions) Ab207442, by Abcam (1 mentions) Ab41621, by Abcam (1 mentions)

31 protocols using molecular weight standard

SDS-PAGE Analysis of Recombinant Human MMP-8

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Example 4

A SDS-PAGE (10%) analysis was performed according to Kiili M et al. (2002).

The recombinant human MMP-8 (Proteaimmun) was activated by APMA and the amounts of rhMMP-8 and the incubation times are indicated below. The bands used for sequencing are shown in FIG. 6.

Lane 1. 1.5 μl molecular weight standard (Bio-Rad)

Lane 2. 2 μl molecular weight standard (Bio-Rad)

Lane 3. 1 μl MMP-8 (0.15 μg/μl)+3 μl TNC buffer (50 mM Tris-HCl, pH 7.8:0.2 M NaCl: 0.75 mM CaCl₂)

Lane 4. empty

Lane 5. 1 μl MMP-8 (0.15 μg/μl)+4 μl 2 mM APMA+3 μl TNC buffer, incubation time 2 h (37° C.)

Lane 6. empty

Lane 7. 1 μl MMP-8 (0.15 μg/μl)+4 μl 2 mM APMA+3 μl TNC buffer, incubation time 5.5 h (37° C.)

The sequencing was performed according to the sequencing method described above. Gel bands 1 to 8 of FIG. 6 were sequenced. The size of the bands were;

Band 2=32 kDa

Band 3=25 kDa

Band 4=21 kDa

Band 5=25 kDa

Band 6=21 kDa

Band 7=12 kDa

Band 8=5 kDa

Bands 3, 4, 5 and 8 comprised SEQ ID NO: 1 and bands 2, 3, 4, 5 and 6 comprised SEQ ID NO: 2. Band 7 identified other fragments of MMP-8. No MMP-8 fragments were identified in band 1. The MMP-8 activation products of band 3, 4 and 5 comprised both SEQ ID NO: 1 and SEQ ID NO: 2. The amino acids 119-132 of SEQ ID NO: 1 and amino acids 151-165 of SEQ ID NO: 2 were from the middle region domain of the total MMP-8 sequence.

US11378579B2. MMP-8 activation product, its determination and use (2022-07-05). OY MEDIX BIOCHEMICA AB [FI], DENTOGNOSTICS GMBH [DE]. Inventors: Timo Sorsa [FI], Dirk-Rolf Gieselmann [DE], Armi Korvuo [FI], Kurt Maier [DE], Païvi Mäntylä [FI], Ismo Råman [FI], Sinikka Tiisala [FI].

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Analytical Size-Exclusion Chromatography

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aSEC was performed using an Agilent HPLC 1100 with a Superdex 200 Increase 5/150 GL column (Cytiva). The system was equilibrated with Tris-buffered saline at pH 7.5 and samples were injected and run at 0.15 ml/min. Absorbance was measured at 280 nm. Molecular weight standards (Biorad) were run separately using identical settings.

Liu W., Maben Z., Wang C., Lindquist K.C., Li M., Rayannavar V., Lopez Armenta I., Nager A., Pascua E., Dominik P.K., Oyen D., Wang H., Roach R.C., Allan C.M., Mosyak L, & Chaparro-Riggers J. (2021). Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex. The Journal of Biological Chemistry, 297(4), 101102.

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Protein Complex Purification by Gel Filtration

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Highly pure individual proteins were chromatographed by gel filtration at 4°C by injecting 2 nmol protein in a 100 µL sample loop onto a Superdex 200 column (24 mL; 10/300 GL, Cytiva Life Sciences) using 20 mM Tris (pH 7.2 at 23°C), 150 mM NaCl, 0.5 mM TCEP as the binding and eluent buffer (0.5 mL/min flow rate). Protein elution was monitored by UV absorbance at 280 nm. The column was calibrated using molecular weight standards (Bio-Rad).
Protein complexes were allowed to form by combining the two components in a 1:1 molar ratio to a final concentration of 20 µM in binding buffer and incubating on ice for 1 hr. Protein complexes were clarified by centrifugation at 16,000 × g for 10 min at 4°C, and 2 nmol of the complexes were immediately injected onto the column. Elution fractions were collected and the fraction corresponding to the CAPN7-IST1 peak was analyzed by Coomassie-stained SDS-PAGE to produce the inset image in Figure 3B.

Paine E.L., Skalicky J.J., Whitby F.G., Mackay D.R., Ullman K.S., Hill C.P, & Sundquist W.I. (2023). The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission. eLife, 12, e84515.

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Quantification of Protein Aggregates

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A Shimadzu UFLC HPLC system equipped with a diode-array detector and a Tosoh Bioscience (Tokyo, Japan) TSK-Gel G3000SW_XL (7.8 mm ID × 30.0 cm, 5μm) and the corresponding guard column (TSK-Gel Guard Column SW_XL, 6.0 mm ID × 4.0 cm, 7μm) were used to monitor for the presence of soluble aggregates (< 100 nm). Prior to sample runs, the columns were rinsed for 60 min with deionized water followed by equilibration at 30°C for 1 h using mobile phase (10 mM sodium phosphate, 450 mM sodium chloride, pH 7.4) at a flow rate of 0.5 ml/min. Molecular weight standards (Biorad Laboratories, Hercules, CA) were run to test for efficacy of separation and resolution. Samples were centrifuged at 16,000 × g for 5 min and 10μL of supernatant was injected for analysis and monitored at 280 nm for each 35 min sample run. Aggregates, monomers, and fragment peaks were quantified using the LC Solutions data analysis software provided with the instrument.

Telikepalli S., Kumru O.S., Kim J.H., Joshi S.B., O'Berry K.B., Blake-Haskins A.W., Perkins M.D., Middaugh C.R, & Volkin D.B. (2014). Characterization of the Physical Stability of a Lyophilized IgG1 mAb After Accelerated Shipping-like Stress. Journal of pharmaceutical sciences, 104(2), 495-507.

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Plasma Fractionation by SEC-HPLC

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Human plasma (100 µL) was fractionated by SEC-HPLC on a Yarra 3 µ SEC-3000 column (300×7.8 mm, Phenomenex, Macclesfield, UK) at 1 ml/min in 50 mM phosphate, NaCl 300 mM, pH 6.8. UV absorption was monitored at 230 nm. The column was calibrated using molecular weight standards (Biorad).

Cauet G., Daynès A, & Temurok N. (2016). Interfering lipoproteins in magnetic field-assisted agglutination of superparamagnetic particles immunoassay. Practical Laboratory Medicine, 4, 82-88.

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SDS-PAGE Analysis of Native and Labeled Ig

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SDS-PAGE was performed under nonreducing conditions with the native or labeled Ig. Proteins (1.5–10 μg per lane) and molecular weight standards (Bio-Rad) were loaded and resolved with a Mini-Protean TGX Precast Gel using a Bio-Rad (Hercules, CA, USA) apparatus. One gel was used for radioactivity measurements using a phosphor screen (Multisensitive Medium MS; Perkin Elmer, Courtaboeuf, France) revealed by an autoradiochromatograph scanner (Cyclone Storage Phosphor System; Perkin Elmer). A duplicate gel was transferred on a PVDF membrane. Membrane was incubated (90 minutes) in diluted primary antibody solution (1/1000, goat antihuman IgG-horse radish peroxidase; Southern Biotech, Birmingham, AL, USA) and revealed with a diaminobenzidine (DAB) substrate.

Carpenet H., Cuvillier A., Monteil J, & Quelven I. (2015). Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations. PLoS ONE, 10(10), e0139835.

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Reagent Procurement for Biochemical Assays

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All chemicals, reagents, enzymes were purchased from Sigma Chemical Company, unless otherwise stated. Molecular weight standards, and protein assay dyes were purchased from BioRad Laboratories.

Gupta S.K, & Crants J. (2019). Identification and impact of stable prognostic biochemical markers for cold-induced sweetening resistance on selection efficiency in potato (Solanum tuberosum L.) breeding programs. PLoS ONE, 14(12), e0225411.

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Purification and Molecular Sizing of BCCIPβ Protein

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Gel filtration was facilitated with the ÄKTA pure chromatography system (GE Healthcare). Purified BCCIPβ-(HIS)₆ (100 μg) was loaded onto a 35 ml Sephacryl S-400 (GE Healthcare) size exclusion chromatography column (1 cm diameter), pre-equilibrated in Buffer B (20 mM KH₂PO₄ pH 7.5, 10% glycerol, 0.5 mM EDTA) with 100 mM KCl. Each fraction (40 fractions total, 1 ml each, 0.5 ml/min flow rate) was precipitated by the addition of deoxycholate (0.02%) and trichloroacetic acid (0.1%), resuspended in 20 μl of SDS loading dye, and resolved using 12% SDS-polyacrylamide gel electrophoresis. The gel was transferred to a nitrocellulose membrane (Whatman) and visualized using HRP-conjugated anti-poly-histidine antibodies (Sigma-Aldrich, A7058; 1:2000) in Buffer F (0.12% 4-chloro-1-naphthol (w/v), 0.12% H₂O₂ (v/v), in PBS). Molecular weight standards (Bio-Rad) were processed in the same manner as above. To establish molecular sizing for the fractions, Molecular weight standards were plotted based on the log of their molecular weight (y-axis) and fraction number (x-axis) using KaleidaGraph 4.1.3, and the linear equation was derived using the linear curve fit tool.

Kelso A.A., Goodson S.D., Watts L.E., Ledford L.L., Waldvogel S.M., Diehl J.N., Shah S.B., Say A.F., White J.D, & Sehorn M.G. (2016). The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing. Nucleic Acids Research, 45(2), 711-725.

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Western Blot Analysis of Protein Expression

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In total, 20 μg of protein of cytosolic fraction from each sample were mixed with loading buffer containing 0,125 M Tris (pH 6.8), 20% glycerol, 10% β-mercaptoethanol, 4% SDS, and 0.002% bromophenol blue, and heated at 50 °C for 5 min. Protein was electrophoresed on 10% SDS–PAGE gel using a miniprotean system (Bio-Rad, Madrid, Spain) with molecular weight standards (Bio-Rad). Protein transfer to nitrocellulose membranes was carried out in the iBlot^TM Dry Blotting System (Invitrogen, Madrid, Spain). Membranes were washed with PBS-Tween 20, blocked with PBS containing 5% skimmed milk, and then incubated with the primary antibodies at 4 °C overnight at 1:1000 dilution for anti-EAAT2 (Abcam, ab41621), 1:600 for anti-AMPK (Abcam, ab207442), 1:1000 for antiphosphorylated AMPK (Abcam, ab23875), and 1:2000 for anti-GAPDH (Abcam, ab8245). GAPDH was used as a gel loading control. After rinsing, the membranes were incubated with the corresponding secondary antibody (Bio-Rad, GAMPO 170-6516, GARPO 172-1019) at a dilution of 1:5000 in PBS containing 5% skimmed milk for 1 h. The antigen was visualized using the ECL chemiluminescence detection kit (Amersham, Madrid, Spain) in a G:Box chamber, and specific bands were quantified by densitometry using GeneTools software (Syngene).

Sánchez-Melgar A., Albasanz J.L., Pallàs M, & Martín M. (2020). Adenosine Metabolism in the Cerebral Cortex from Several Mice Models during Aging. International Journal of Molecular Sciences, 21(19), 7300.

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Purification and Characterization of hMCM10

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Wild type or ΔN27 hMCM10-ID (200 µL at 5 mg ml⁻¹) was injected into a Superdex 75 Increase 10/300 GL column (GE Healthcare) operating with the elution buffer containing 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5 mM β-mercaptoethanol, and a flow rate of 0.4 mL min⁻¹. The proteins were detected by UV absorption at 280 nm. To calibrate the column, we injected molecular weight standards (Bio-Rad) including thyroglobulin, γ-globulin, ovalbumin, myoglobin, and vitamin B12 under the same elution condition. All experiments were performed at 4 °C.

Baxley R.M., Leung W., Schmit M.M., Matson J.P., Yin L., Oram M.K., Wang L., Taylor J., Hedberg J., Rogers C.B., Harvey A.J., Basu D., Taylor J.C., Pagnamenta A.T., Dreau H., Craft J., Ormondroyd E., Watkins H., Hendrickson E.A., Mace E.M., Orange J.S., Aihara H., Stewart G.S., Blair E., Cook J.G, & Bielinsky A.K. (2021). Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening. Nature Communications, 12, 1626.

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