Log In Sign up
The largest database of trusted experimental protocols

Gradient sds page

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States

The 4–12% gradient SDS-PAGE is a laboratory equipment used for the separation and analysis of proteins. It is a type of polyacrylamide gel electrophoresis (PAGE) that utilizes a sodium dodecyl sulfate (SDS) buffer system to denature proteins and separate them based on their molecular weight.

Automatically generated - may contain errors

Lab products found in correlation

Protease inhibitor, by Merck Group (2 mentions) Ecl plus system, by Cytiva (2 mentions) Pdgf c, by R&D Systems (2 mentions) Hrp conjugated secondary antibody, by Zymo Research (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Ecl plus kit, by Thermo Fisher Scientific (2 mentions) Blocking buffer, by LI COR (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Antibodies against human p53 phosphorylated at ser15, by Cell Signaling Technology (2 mentions) Np40 cell lysis buffer, by Thermo Fisher Scientific (2 mentions) Supersignal west femto substrate, by Thermo Fisher Scientific (2 mentions) Anti β actin clone ac 15, by Merck Group (1 mentions) Protease inhibitor, by Roche (1 mentions) Anti flag m2 magnetic bead, by Merck Group (1 mentions) 3xflag peptide, by Merck Group (1 mentions) Pierce silver stain for mass spectrometry, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Pcmv6 entry vector, by OriGene (1 mentions)

20 protocols using gradient sds page

1

Quantifying Ubiquitin Modification in Hippocampal Slices

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
Organotypic hippocampal slices (6 slices pooled/sample) were removed from Millicell inserts with a sterile metal scraper and homogenized with ice-cold homogenization buffer (15mM Tris-HCl pH 7.6, 250mM sucrose, 1mM MgCl2, 2.5mM EDTA, 1mM EGTA, 1mM Na3VO4, 5mM NaF, 20mM phenylphosphate, 20mM N-ethylmaleimide, 1mM DTT, 1x protease inhibitors (Roche Applied Sciences, Indianapolis, IN)). Triton X100-resistant proteins were isolated by sequential centrifugation with increasing Triton X100 and KCl concentrations as previously published [26 (link),39 (link),40 (link)]. Fysates were mixed with SDS sample buffer (62mM Tris-HCl pH6.8, 10% glycerol, 2% SDS, bromophenol blue, 50mM DTT), loaded on 4-12% gradient SDS-PAGE (Thermo Fisher Scientific) and screened for the absence and presence of high molecular weight ubiquitin by Western Blotting with anti-ubiquitin antibody (clone ubi-1, Thermo Fisher Scientific), as well as anti-ubiquitin Lys48- (clone Apu2, EMD Millipore) and Lys63-(clone D7A11, Cell Signaling Technology, Danvers, MA) specific antibodies. GluN1, Shank and β-actin protein levels were assessed with anti-GluN1 (clone N308/48, BioLegend, San Diego, CA), anti-pan Shank (clone N23B/49, BioLegend) and anti-β-actin (clone AC-15, Sigma Aldrich) antibodies.
Kahles T., Poon C., Qian L., Palfini V., Srinivasan S.P., Swaminathan S., Blanco I., Rodney-Sandy R., Iadecola C., Zhou P, & Hochrainer K. (2020). Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition. Cellular and molecular life sciences : CMLS, 78(5), 2169-2183.
+ Open protocol
+ Expand
2

SARS-CoV-2 S Protein Immunoprecipitation

Check if the same lab product or an alternative is used in the 5 most similar protocols
The HCoV-OC43-infected cell lysates were incubated with anti-HCoV-OC43 S Ab for 3 h at 4°C. Protein A beads (CaptivA™ PriMAB 52% (v/v) slurry, Repligen, Waltham, MA, USA) were added into the cell lysates. After 1 h incubation at 4°C, the immunocomplexes were collected by centrifugation, then separated by 4–12% gradient SDS-PAGE (Thermo Fisher Scientific) and subjected to western blotting analysis using either anti-HCoV-OC43 N monoclonal Ab or anti-HCoV-OC43 S Ab.
Kim J., Kim M., Kim D., Park S., Kang M., Baek K., Choi J.K., Maharjan S., Akauliya M., Lee Y, & Kwon H.J. (2022). Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43. Frontiers in Immunology, 13, 835333.
+ Open protocol
+ Expand
3

Affinity Purification of LZTFL1 Complexes

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
Jurkat E6.1 cell line was cultured in RPMI-1640 (Thermo Fisher Scientific) as previously described [21 (link)] and transfected with plasmids expressing LZTFL1-Myc-Flag or pCMV6-Entry vector (Origene) by electroporation (BioRad) and cultured for 48 hours. The FLAG-fusion proteins were immunoprecipitated by incubating with anti-FLAG M2 magnetic beads (Sigma) overnight at 4°C. The beads were washed three times with lysis buffer, and the bound proteins were eluted with 3X FLAG peptide (Sigma). Eluted proteins were incubated with NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 99°C for 10 minutes then separated on 4–12% gradient SDS-PAGE (Thermo Fisher Scientific).
Protein gel was stained with Pierce silver stain for mass spectrometry (Thermo Fisher Scientific) according to the manufacturer’s protocol. In-gel digestion and mass spectrometry analysis were carried out as previously described [22 (link)].
Promchan K, & Natarajan V. (2020). Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1. PLoS ONE, 15(1), e0226298.
+ Open protocol
+ Expand
4

Proteomic Analysis of Cerebral Cortex

Check if the same lab product or an alternative is used in the 5 most similar protocols
Whole-cell protein extracts were obtained from the ipsilateral cerebral cortices (+1 to −4 mm from bregma). Brain tissues were homogenized in cold RIPA buffer (50 mM Tris-HCI with pH 8.0, 150 mM NaCI, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1% NP-40) containing protease inhibitor (Pierce tablet; Thermo Fisher Scientific, A32965) and phosphatase inhibitor (Sigma, P0044). The homogenates were centrifuged at 13,000 rpm for 20 min at 4°C, and the supernatants were used for analysis. Protein content was measured using Bradford dye reagent (Bio-Rad, Hercules, CA). Protein samples (30 μg protein per sample) were separated by 4-20% gradient SDS-PAGE (Invitrogen) and transferred to PVDF membranes. The following primary antibodies were used: rabbit anti-LC3B (1:2000; Novus Biological, NB100-2220), rabbit anti-p62 (1:1000), mouse anti-CTSD (1:100),mouse anti-GAPDH (1:10000; Sigma, G8795), rabbit anti-postsynaptic density 95 (PSD-95; 1:1000; Cell Signaling, 3409), and mouse anti-β-actin (1:800; Sigma, A2066). Immunopositive bands of horseradish peroxidase-conjugated secondary antibodies were detected with an ECL system (GE Healthcare) and exposure to ECL Hyperfilm.
Jalin A.M., Jin R., Wang M, & Li G. (2019). EPPS Treatment Attenuates Traumatic Brain Injury in Mice by Reducing Aβ Burden and Ameliorating Neuronal Autophagic Flux. Experimental neurology, 314, 20-33.
+ Open protocol
+ Expand
5

Serum Albumin Comparative Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Albumins from mouse, bovine and human serum were boiled at 85°C for 2 minutes, fractionated in pre-casted 4–20% gradient SDS-PAGE (Invitrogen), and stained with Coomassie G-250 Blue (Invitrogen), Replicate gels were used for Western blot analysis, using 0.45µm nitrocellulose membranes. Membranes were blocked with 2% OVA in PBS for 1 hour and incubated overnight with 1/100 dilution of pooled mouse sera diluted in 2% OVA. Mouse antibodies were detected by incubation with polyclonal goat anti-mouse IgG (H+L)-HRP (Bio-Rad), followed by colorimetric development using 4-chloronaphtol as substrate.
Colino J., Duke L, & Snapper C.M. (2014). Autologous albumin enhances the humoral immune response to capsular polysaccharide covalently co-attached to bacteria-sized latex beads. European journal of immunology, 44(5), 1433-1443.
+ Open protocol
+ Expand
6

Protein-Protein Interaction Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cultured cells (A549 and D562) and bacteria (E. coli or Spn WU2) were lysed by sonication in RIPA buffer containing 1X protease inhibitors (Sigma). Cell lysates (20 μg), bacterial lysates (20 μg) or 2 μg of recombinant huGAPDH were mixed with 2 μg of 6His-PspA or 6His-PspA fragments on buffer A (PBS, 0.1% Triton X-100, 0.5 mM DTT and 10 mM imidazole) and then incubated for 1hourat 4°C on rotator (6 rpm). Ni-NTA resin (20 μl) was equilibrated in buffer A. Subsequently, Ni-NTA resin and mixtures of proteins were mixed together and incubated for 1 hour at 4°C on a rotator (6 rpm). Incubated Ni-NTA resins were washed four times with 1 mL of buffer A containing 20 mM imidazole at 2,000 X g. 2X SDS loading buffer was added to the Ni-NTA resins and then incubated at 90°C for 5 min. Proteins were separated on 4%–12% gradient SDS-PAGE (Invitrogen) and visualized by Coomassie blue staining or immunoblots. For protein identification, the Coomassie blue stained bands were cut out of the gel and digested with trypsin overnight. Elution of the peptides were identified using LC-MS at the Cancer Center at University of Alabama at Birmingham, USA.
Park S.S., Gonzalez-Juarbe N., Riegler A.N., Im H., Hale Y., Platt M.P., Croney C., Briles D.E, & Orihuela C.J. (2021). Streptococcus pneumoniae binds to host GAPDH on dying lung epithelial cells worsening secondary infection following influenza. Cell reports, 35(11), 109267.
+ Open protocol
+ Expand
7

Quantification of Anti-F1 Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols
Serum proteins (10 μl) were separated by 4–12% gradient SDS-PAGE (Invitrogen) and were stained using a Silver Stain Plus kit (Bio-Rad). For Western blot analysis, electrophoresed samples were transferred to nitrocellulose membranes (Invitrogen). Membranes were developed with polyclonal antibodies against hemopexin (Abcam ab 90947), transferrin (Abcam ab 82411) and β-actin (Sigma A5060), followed by HRP-conjugated goat anti-rabbit IgG. Anti-F1 ELISA in the serum of immunized mice was performed as described previously (Levy et al., 2011 (link), 2016 (link)). Briefly, microtiter plates were coated with 500 ng of purified rF1 [provided by the Biotechnology Department at IIBR, produced as described in Holtzman et al. (2006 (link))]. Tested sera were serially diluted in 2-fold dilutions in a final volume of 50 μl and were incubated in the wells for 1 h at 37°C. Alkaline phosphatase-conjugated goat anti-rabbit IgG (1/2,000 dilution, Sigma) was used as the 2nd layer for rabbit anti-F1 IgG titer determination. Titers were defined as the reciprocal values of the endpoint serum dilutions that displayed OD405 values 2-fold higher than the normal serum controls.
Zauberman A., Vagima Y., Tidhar A., Aftalion M., Gur D., Rotem S., Chitlaru T., Levy Y, & Mamroud E. (2017). Host Iron Nutritional Immunity Induced by a Live Yersinia pestis Vaccine Strain Is Associated with Immediate Protection against Plague. Frontiers in Cellular and Infection Microbiology, 7, 277.
+ Open protocol
+ Expand
8

PDGF-C Protein Expression in Mouse Retina

Check if the same lab product or an alternative is used in the 5 most similar protocols
Preparation of whole cell lysates and Western blot analysis were performed as described previously.42 (link) Cell extracts from mouse retina (30 µg/lane) were fractionated on 4–12% gradient SDS-PAGE (Invitrogen). Antibodies used included PDGF-C (R&D Systems, Minneapolis, MN, USA) and β-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Preimmune serum was used in parallel as controls and signals were detected with a HRP-conjugated secondary antibody (Zymed Laboratories) using the ECL-PLUS system (Amersham, Arlington Heights, IL, USA). Assays were repeated in 4 WT and 5 DKO mice. Image-J software is used to measure the band intensity in pixels.
Wang Y., Abu-Asab M.S., Yu C.R., Tang Z., Shen D., Tuo J., Li X, & Chan C.C. (2014). Platelet-derived growth factor (PDGF)-C inhibits neuroretinal apoptosis in a murine model of focal retinal degeneration. Laboratory investigation; a journal of technical methods and pathology, 94(6), 674-682.
+ Open protocol
+ Expand
9

Western Blot Analysis of eIF2α and HSP70

Check if the same lab product or an alternative is used in the 5 most similar protocols
Twenty-five-day heat-treated male fly whole bodies or heads were homogenized in 4× lithium dodecyl sulfate (LDS) loading buffer and 10× sample reducing agent (Thermo Fisher Scientific, Waltham, MA, USA) for Western blot analysis. To separate the total protein extract, 4–12% gradient SDS-PAGE (Invitrogen, Carlsbad, CA, USA) was used, which was transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The primary antibodies used were as follows: total eIF2α (1:200; Abcam, Cambridge, UK, ab26197-100), phosphor-eIF2α (1:1000; Cell Signaling, Danvers, MA, USA, 9721S), β-actin (1:5000; Cell Signaling, Danvers, MA, USA, 4967S), or HSP70 (1:1000; Enzo Life Sciences, Farmingdale, NY, USA, SPA-822). Secondary antibodies used were as follows: goat anti-rabbit IgG horseradish peroxidase (HRP) and goat anti-mouse IgG HRP conjugate (1:2000; Millipore, Burlington, MA, USA, AP307P, AP308P). Proteins were detected using an ECL-Plus kit (Thermo Fisher Scientific, Waltham, MA, USA).
Elvira R., Cha S.J., Noh G.M., Kim K, & Han J. (2020). PERK-Mediated eIF2α Phosphorylation Contributes to The Protection of Dopaminergic Neurons from Chronic Heat Stress in Drosophila. International Journal of Molecular Sciences, 21(3), 845.
+ Open protocol
+ Expand
10

Western Blot Protein Detection Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell lysates were prepared by disrupting cell pellets in the Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) supplemented with 5% beta-mercaptoethanol (Sigma-Aldrich), and heated at 96°C for 5 min. Samples were then subjected in duplicate to 4–12% gradient SDS-PAGE (Invitrogen) and transferred onto PVDF membranes using BioRad semi-dry transblotters. Membranes were blocked with LiCor blocking buffer (LiCor, Lincoln, NE) for 1 h at room temperature, and probed with primary antibodies (described in Supplementary Table 1) diluted in LiCor blocking buffer containing 0.1% Tween. The membranes were washed twice for 10 min each in PBS with 0.1% Tween (PBST) and then probed with goat anti-mouse or goat anti-rabbit IR-Dye 670- or 800 cw-labeled secondary antisera diluted in LiCor blocking buffer containing 0.1% Tween and 0.01% SDS for 1 h at room temperature. The probed membranes were washed twice for 10 min in TBST, placed in water, and imaged using a LiCor Odyssey scanner. Boxes were manually placed around each band of interest, and near-infrared fluorescent values for raw intensity, with intra-lane background subtracted, were obtained using Odyssey 3.0 analytical software (LiCor).
Nakamura K., Nakayama K., Ishikawa N., Ishikawa M., Sultana R., Kiyono T, & Kyo S. (2017). Reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells. Oncotarget, 9(16), 12609-12619.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!