Seeblue plus2 protein standard

Manufactured by Thermo Fisher Scientific

Sourced in United States

SeeBlue® Plus2 Protein Standard is a pre-stained protein ladder used to estimate the molecular weight of proteins during SDS-PAGE analysis. It contains a mixture of proteins with a known molecular weight range.

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Lab products found in correlation

Anti lamin a c, by Abcam (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Anti rabbit secondary antibody, by Merck Group (1 mentions) Anti actin, by Merck Group (1 mentions) Chemicon, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Illustra tripleprep kit, by GE Healthcare (1 mentions) Image studio software version 5, by LI COR (1 mentions) Protease inhibitor tablet, by Roche (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Odyssey fc imaging system, by LI COR (1 mentions) Mouse anti tubulin, by Merck Group (1 mentions) Protran, by Cytiva (1 mentions) Submarine system, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Mops sds running buffer, by Thermo Fisher Scientific (1 mentions) Sharp pre stained protein standard, by Thermo Fisher Scientific (1 mentions) Imperial protein stain, by Thermo Fisher Scientific (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions)

Spelling variants of this product

SeeBlue Plus2 Pre-stained Protein Standard (69 citations) SeeBlue Plus2 (64 citations) SeeBlue (16 citations) Protein standards (14 citations) SeeBlue Plus2 Protein Standard 1X (2 citations)

8 protocols using seeblue plus2 protein standard

Protein Extraction and Western Blot Analysis

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The sample pellets were diluted in 150–300 μl of 1× phosphate buffered saline with a Protease inhibitor cocktail (Sigma-Aldrich) and sonicated. A NuPAGE^® LDS Sample Buffer (Life Technologies) and 5 mM DTT were added and the samples were subjected to the 30 min incubation at 50°C. Alternatively, total proteins were extracted using the Illustra Triple Prep Kit (GE Healthcare Life Sciences). The western blot was carried out according to a standard procedure using pre-cast gel cassettes (Life Technologies). We used anti-ZNF555 (Ab4) (Sigma-Aldrich), anti-Actin (Sigma-Aldrich) and anti-Lamin A/C (Abcam) antibodies in 1/1000 dilution and anti-rabbit secondary antibodies (Sigma-Aldrich) in 1/1000 dilution. The SeeBlue Plus2 Protein Standard (Life Technologies) was used to detect the size of analyzed protein. The detection solutions were the SuperSignal West Pico Chemiluminescent Substrate (Pierce Thermo Scientific) or Chemicon (Millipore).

Kim E., Rich J., Karoutas A., Tarlykov P., Cochet E., Malysheva D., Mamchaoui K., Ogryzko V, & Pirozhkova I. (2015). ZNF555 protein binds to transcriptional activator site of 4qA allele and ANT1: potential implication in Facioscapulohumeral dystrophy. Nucleic Acids Research, 43(17), 8227-8242.

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Western Blot Analysis of HDAC6 in Mouse Lung

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Protein was extracted from the homogenised lung tissue using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4 and 1× protease inhibitor tablet (Roche, Basel, Switzerland)), and total protein concentration was determined by the Pierce™ BCA protein assay kit (ThermoFisher Scientific, MA, USA). An equal amount (50 μg) of protein from WT and KO mouse lung tissue was resolved on 8% Tris-Glycine SDS-PAGE along with SeeBlue^® Plus 2 protein standard (Life Technologies). The resolved polypeptides were transferred onto a Protran^® Premium nitrocellulose membrane (GE Healthcare, IL, USA). The membrane was blocked with 5% non-fat dry milk, and probed with the rabbit anti-HDAC6 (D21B10, Cell Signalling, MA, USA) or mouse anti-tubulin (Sigma-Aldrich) antibody, followed by the horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Life Technologies) IgG. The protein bands were visualised using ECL or ECL Prime Western Blotting Systems (GE Healthcare), and the images were acquired on Odyssey Fc imaging system with Image Studio software version 5.0 (Li-COR, NE, USA) and exported as TIFF images.

Zanin M., DeBeauchamp J., Vangala G., Webby R.J, & Husain M. (2020). Histone Deacetylase 6 Knockout Mice Exhibit Higher Susceptibility to Influenza A Virus Infection. Viruses, 12(7), 728.

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SDS-PAGE, Protein Transfer, and Immunostaining

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Samples were size-separated by SDS-PAGE using Novex 4-12% BIS-TRIS gradient gels and MOPS SDS running buffer (Life Technologies), or homemade 8% acrylamide gels and TRIS-glycine running buffer. Subsequently, proteins were transferred to nitrocellulose membranes (Amersham Biosciences) using a submarine system (Life Technologies). Sharp Pre-stained Protein Standard (Novex) was used as a size indicator for figure 1, and SeeBlue® Plus2 Protein Standard (Life Technologies) was used as a size indicator for figures 2-6. Note that SeeBlue® Plus2 migrates differently depending on buffer conditions used, as documented on the manufacturer’s website. Ponceau-S staining and immunostaining were performed as described previously (38 (link)).

Hendriks I.A., Schimmel J., Eifler K., Olsen J.V, & Vertegaal A.C. (2015). Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4). The Journal of Biological Chemistry, 290(25), 15526-15537.

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Protein Visualization by SDS-PAGE

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Changes in proteins over time during incubation with pepsin, trypsin, and chymotrypsin were visualized with reducing SDS-PAGE. Laemmli sample buffer and 10× tris/glycine/SDS running buffer were obtained from Bio-Rad (Hercules, CA), β-mercaptoethanol was obtained from Sigma-Aldrich, and SeeBlue Plus2 protein standard was obtained from Invitrogen (Carlsbad, CA). Sample and sample buffer (containing 1% β-mercaptoethanol) were combined in a 1:1 ratio and the mixture was heated for 5 min at 85°C. Samples were run on a 15% tris-HCl Ready Gel (Bio-Rad Laboratories) at constant voltage (200 V). Gels were stained with Imperial Protein Stain (Thermo Fisher Scientific, Rockford, IL). Band density was quantified with UN-SCAN-IT gel analysis software (Silk Scientific, Inc., Orem, UT).

Maloney K.P., Truong V.D, & Allen J.C. (2014). Susceptibility of sweet potato (Ipomoea batatas) peel proteins to digestive enzymes. Food Science & Nutrition, 2(4), 351-360.

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Protocol for Selective Digestion of HMM

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HMM was selectively digested at the actin-binding loop 2 (cleaved HMM) as described in Bobkov et al. (1996) (link) with minor modifications. Briefly, HMM was dialyzed against 0.02 M KCl, 0.02 M Tris–HCl and 1 mM DTT at pH 7.4. To protect loop 1 from digestion, the ionic strength was increased to 0.5 M KCl and ATP was added to 6 mM (Mocz et al. 1984 (link)). TPCK-treated trypsin (proteolytic activity 0.08 BTEE units/mg; Sigma-Aldrich T1426) was added to HMM to a final concentration of 0.05 mg/ml and incubated on ice for 6 min. Digestion was stopped using soybean trypsin inhibitor type 1-S (Sigma-Aldrich T9003) at a molar ratio of 3:1 to trypsin. Cleaved HMM was used within 48 h of digestion, and then discarded.
Digestion was assessed by electrophoresis on NuPAGE 12 % Bis–Tris Gel in MOPS running buffer and gels were stained using SimplyBlue^™ Safe Stain (Invitrogen, Carlsbad, CA). The molecular weight of fragments was determined by comparison to SeeBlue^® Plus2 Protein Standard (Invitrogen) using the AlphaEaseFC (Alpha Innotech Corp., San Leandro, CA) molecular weight tool. As in previous experiments digestion of HMM mainly resulted in two fragments, the N-terminal fragment of S1 and the combined C-terminal region of S1 fragment and S2 (Bálint et al. 1975 (link); Bobkov et al. 1996 (link)) (Fig. 1). Other contaminating fragments were present in low amounts.

Clobes A.M, & Guilford W.H. (2014). Loop 2 of myosin is a force-dependent inhibitor of the rigor bond. Journal of muscle research and cell motility, 35(2), 143-152.

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Western Blot Analysis of Drosophila Seminal Proteins

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Proteins were extracted from 3–4 day old virgin male AG or heads (as in [38 (link)]). Western blotting for Acp36DE, Ovulin, Acp62F, sex peptide (SP), and Actin was performed as previously described [39 (link)]. Western blotting for Rh1 was as previously described [40 (link)]. The Rh1 monoclonal antibody (4C5) was purchased from the Developmental Studies Hybridoma Bank (University of Iowa). The size markers in Fig. 2A and Fig. 3D are from the SeeBlue Plus2 Protein Standard (Invitrogen). All Western blot analyses were performed in triplicate independent experiment. Representative images are shown in the figures.

Chow C.Y., Avila F.W., Clark A.G, & Wolfner M.F. (2015). Induction of Excessive Endoplasmic Reticulum Stress in the Drosophila Male Accessory Gland Results in Infertility. PLoS ONE, 10(3), e0119386.

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Cloning and Expression of Recombinant Proteins

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The pET30a(+) vector was obtained from Novagen (Madison, WI). Synthetic oligonucleotides were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes, calf intestinal phosphatase (CIP), T4 ligase, and Quick ligation kits were purchased from New England Biolabs (Beverley, MA). DNA purification was performed using kits from Qiagen (Valencia, CA). Ninitrilotriacetic acid (Ni-NTA)-agarose resin for protein purification was acquired from Qiagen (Valencia, CA). Slide-A-Lyzer dialysis cassettes were obtained from Pierce (Rockford, IL). The 1kb DNA ladder, NuPAGE 4–12% Bis-tris protein gels, SeeBlue Plus 2 protein standard, and T1 phage-resistant cells were purchased from Invitrogen (Carlsbad, CA). All cloning steps were performed in Escherichia coli strain DH5a from Invitrogen (Carlsbad, CA). Protein expression was carried out in E. coli strain BL21DE3 (Invitrogen, Carlsbad, CA). Kanamicine and imidazole were purchased from Sigma (St. Louis, MO). Silicon wafer chips were obtained from Ted Pella, Inc. (Redding, CA). Other chemicals with the highest grade of purity were purchased from Fisher Scientific (Pittsburg, PA).

Tokareva O.S., Lin S., Jacobsen M.M., Huang W., Rizzo D., Li D., Simon M., Staii C., Cebe P., Wong J.Y., Buehler M.J, & Kaplan D.L. (2014). Effect of Sequence Features on Assembly of Spider Silk Block Copolymers. Journal of structural biology, 186(3), 412-419.

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Secreted Protein Profiling of aEPEC Strains

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To perform the secreted protein profile of each aEPEC strain we first selected single colonies of wild-type strains, which were inoculated in LB broth and cultured at 37°C for 18–24 h. Each culture was then sub-cultured into fresh preheated DMEM medium (2 g/L sodium bicarbonate, 4.5 g/L glucose) at a dilution of 1/50 and incubated at 37°C with constant rotation (250 rpm) until they reached an OD₆₀₀ ≅ 1. After 5 h at 37°C under continuous agitation (250 rpm), the secreted proteins contained in the supernatants of these cultures were then precipitated with trichloroacetic acid (10% TCA) and resolved by 12% SDS-PAGE. SeeBlue^® Plus2 Protein Standard (Invitrogen, CA, United States) was used as a molecular weight marker. Protein extracts were electrophoresed at 150 V for 1 h and 30 min. Subsequently, the gel was washed 3 times with distilled water for 5 min to remove excess SDS. To visualize the secreted protein profile, the gel was stained for 1 h under gentle agitation with Coomassie Brilliant Blue R-250 (BioRad, CA, United States) and subsequently rinsed for 30 min in distilled water.

Santos F.F., Yamamoto D., Abe C.M., Bryant J.A., Hernandes R.T., Kitamura F.C., Castro F.S., Valiatti T.B., Piazza R.M., Elias W.P., Henderson I.R, & Gomes T.A. (2019). The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic Escherichia coli (aEPEC) Can Mediate Adherence. Frontiers in Microbiology, 10, 1527.

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