Un scan it gel version 6.1 scanning software

Manufactured by Silk Scientific

Sourced in United States

Un-Scan-It gel version 6.1 is a software application that allows users to digitize and analyze gel electrophoresis images. The software provides basic functionalities for image capture, processing, and quantification of bands or spots within the gel.

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Lab products found in correlation

Wbkls0500, by Merck Group (7 mentions) Bca protein assay, by Thermo Fisher Scientific (6 mentions) Crmp2, by Merck Group (4 mentions) Nupage gel, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Gsk 3β pser9, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence western blotting substrate, by Thermo Fisher Scientific (1 mentions) Gsk3β, by Cell Signaling Technology (1 mentions) Actin, by Merck Group (1 mentions) Amershamtm protrantm, by GE Healthcare (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Novex gel, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions) Benzonase, by Merck Group (1 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions) βiii tubulin, by Promega (1 mentions)

9 protocols using un scan it gel version 6.1 scanning software

Western Blot Analysis of Neuronal Proteins

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Indicated samples were loaded on 4–12% NuPAGE® gels (Life Technologies). Proteins were transferred to polyvinylidene difluoride membranes preactivated in methanol and blocked at room temperature for 1 h with TBST containing 5% non-fat dry milk. The saturated membranes were incubated separately in TBST containing 5% BSA with the primary antibodies CRMP2 (Cat# C2993, Sigma, St. Louis, MO, USA), NR2B (Cat# 610416, BD biosciences, San Jose, CA, USA) or Kv2.1 (Cat# K89/34, NeuroMab, Davis, CA, USA) overnight at 4°C. Following incubation in horseradish peroxidase conjugated secondary antibodies (Jackson immunoresearch), blots were revealed by enhanced luminescence (WBKLS0500, Millipore) before exposure to photographic film. Films were scanned, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc, Orem, UT, USA).

Moutal A., François-Moutal L., Brittain J.M., Khanna M, & Khanna R. (2015). Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides. Frontiers in Cellular Neuroscience, 8, 471.

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Western Blot Analysis of Cortical Neuron Lysates

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Lysates were made from cultured cortical neurons at 6 DIV in RIPA buffer containing protease and phosphatase inhibitor cocktails as previously described (Brittain et al., 2009 (link)). Samples were boiled in Laemmli sample buffer and separated by electrophoresis on SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and blocked at room temperature for 1 h and incubated in primary antibodies CRMP2 (Cat# C2993, Sigma, St. Louis, MO), CRMP2 pThr509/Thr514 (Cat# PB-043, Kinasource, Dundee, Scotland, UK), CRMP2 pSer522 (Cat# CP2191, ECM Biosciences, Versailles, KY), GSK3β pSer9 (Cat# 5558, Cell Signaling, Danvers, MA), Cdk5 (Cat# 2506, Cell Signaling, Danvers, MA), p35 (Cat# 2680, Cell Signaling, Danvers, MA), GSK3β (Cat# 9832, Cell Signaling, Danvers, MA), or Tubulin (Cat# G712A, Promega, Madison, WI) overnight at 4°C. Following incubation in horseradish peroxidase conjugated secondary antibodies, blots were probed with enhanced chemiluminescence Western blotting substrate (Thermo Scientific, Waltham, MA) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc, Orem, UT).

Wilson S.M., Moutal A., Melemedjian O.K., Wang Y., Ju W., François-Moutal L., Khanna M, & Khanna R. (2014). The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth. Frontiers in Cellular Neuroscience, 8, 196.

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Examining LCM Effect on CRMP2 Phosphorylation

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For examining the effect of (S)-LCM on CRMP2 phosphorylation state, TGs were dissected from adult rats and treated for 30 min at 37°C with 200 µM (S)-LCM diluted in Dulbecco’s modified essential media (DMEM; Cat#11965, Life technologies). Next, tissues were lysed by sonication in RIPA buffer (50mM Tris-HCl, pH 7.4, 50mM NaCl, 2mM MgCl₂, 1% [vol/vol] NP40, 0.5% [mass/vol] sodium deoxycholate, and 0.1% [mass/vol] sodium dodecyl sulfate and protease (Cat#B14002; Biotool) and phosphatase inhibitors (Cat#B15002, Biotool), and BitNuclease (Cat#B16002, Biotool)). Protein concentrations were determined using the BCA protein assay (Cat#PI23225, Thermo Fisher Scientific). Approximately 5µg of total proteins were loaded on an SDS-PAGE and then transferred to polyvinylidene difluoride membranes and blocked at room temperature for 1 hour in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1 % Tween 20), 5% non-fat dry milk. Primary antibodies used for probing were CRMP2 (Cat#C2993, Sigma, St Louis, MO), CRMP2 pSer522 (Cat#CP2191, ECM Biosciences), and Actin (Cat#A2066, Sigma, St Louis, MO) and were diluted in TBST with 5% bovine serum albumin. Immunoblots were revealed by enhanced luminescence (Cat#WBKLS0500, Millipore) before exposure to a photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc).

Moutal A., Eyde N., Telemi E., Park K.D., Xie J.Y., Dodick D.W., Porreca F, & Khanna R. (2016). Efficacy of (S)-lacosamide in preclinical models of cephalic pain. Pain Reports, 1(1), e565.

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Protein Expression Analysis by Western Blot

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Total protein (10 μg) from the tissue supernatant was loaded on TGX precast gels (10% Criterion^TM, BioRad) and then transferred to nitrocellulose membranes (Amersham^TM Protran^TM, GE Healthcare). After transfer, the membranes were blocked at room temperature for 1 h in blocking buffer (5% BSA in Tris-buffered saline with Tween 20 (TBST)). The following primary antibodies were diluted in blocking buffer: NHE1 (Abcam, ab67313, 1:500), and α-tubulin (Cell Signaling, 3873S, 1:20,000). The membranes were incubated in the diluted primary antibodies overnight at 4°C. The blots were then washed three times in TBST, incubated with secondary antibodies: GαM680 (LiCor, 926–68020), and GαR800 (LiCor, 926–32211) in 5% milk in TBST for 1 h of rocking at room temperature, washed again, and imaged with a LiCor Odyssey infrared imaging system (LiCor, Lincoln, NE). Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc.) was used for quantification. To visualize multiple bands on the same blot, blots were stripped with One Minute® Plus Western Blot Stripping Buffer (GM Biosciences, GM6510). Protein expressions were corrected for the expression of the loading control (e.g. α-tubulin).

Liktor-Busa E., Blawn K.T., Kellohen K.L., Wiese B.M., Verkhovsky V., Wahl J., Vivek A., Palomino S.M., Davis T.P., Vanderah T.W, & Largent-Milnes T.M. (2020). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake. PLoS ONE, 15(5), e0227463.

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Affinity Purification of CRMP2 Protein Complexes

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Glutathione magnetic beads (Cat# B23702, Biotools), preincubated with purified CRMP2-GST (~0.4 µM), were incubated overnight with 300 µg of total protein from spinal cord lysates at 4˚C in the absence or presence of the indicated peptides with gentle rotation. Beads were washed 3 times with lysis buffer before resuspension in Laemmli buffer and denaturation (5 minutes at 95˚C) and immunoblotting as described previously.^{4 (link),36 ,83 (link)} Immunoblots were revealed by enhanced luminescence (WBKLS0500, Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc, Orem, UT).

Moutal A., Wang Y., Yang X., Ji Y., Luo S., Dorame A., Bellampalli S.S., Chew L.A., Cai S., Dustrude E.T., Keener J.E., Marty M.T., Vanderah T.W, & Khanna R. (2017). Dissecting the role of the CRMP2–neurofibromin complex on pain behaviors. Pain, 158(11), 2203-2221.

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Western Blot Protein Detection

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Indicated samples were loaded on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific). Proteins were transferred for 1 hour at 120 V using TGS (25 mM Tris pH = 8.5, 192 mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to polyvinylidene difluoride membranes 0.45 µm (Cat# IPVH00010; Millipore), preactivated in pure methanol. After transfer, the membranes were blocked at RT for 1 hour with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), 5% (mass/vol) nonfat dry milk, then incubated separately in indicated primary antibodies in TBST, 5% (mass/vol) BSA, overnight at 4˚C. Following incubation in horseradish peroxidase-conjugated secondary antibodies from Jackson immunoresearch, blots were revealed by enhanced luminescence (WBKLS0500; Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software by Silk Scientific Inc.

Moutal A., Yang X., Li W., Gilbraith K.B., Luo S., Cai S., François-Moutal L., Chew L.A., Yeon S.K., Bellampalli S.S., Qu C., Xie J.Y., Ibrahim M.M., Khanna M., Park K.D., Porreca F, & Khanna R. (2017). CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide. Pain, 158(12), 2301-2319.

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CRMP2 Interaction Assay in Rat Spinal Cord

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Lumbar segment of spinal cord lysates prepared from adult Sprague Dawley rats were generated by homogenization and sonication in RIPA buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2 mM MgCl₂, 1% (vol/vol) NP40, 0.5% (mass/vol) sodium deoxycholate, 0.1% (mass/vol) SDS). The RIPA buffer included freshly added protease inhibitors (Cat# B14002, Biotools, Houston, TX), phosphatase inhibitors (Cat# B15002, Biotools, Houston, TX), and Benzonase (Cat# 71206, Millipore, Billerica, MA). Protein concentrations were determined using the BCA protein assay (Cat# PI23225, Thermo scientific). Glutathione magnetic beads (Cat# B23702, Biotools, Houston, TX), pre-incubated with purified CRMP2-GST (~0.4 μM), were incubated overnight with 300 μg of total protein from spinal cord lysates at 4°C in the absence or presence of the indicated peptides with gentle rotation. Beads were washed 3 times with RIPA buffer before re-suspension in Laemmli buffer and denaturation (5 min at 95°C) and immunoblotting [12 (link); 42 (link); 90 (link)]. Immunoblots were revealed by enhanced luminescence (WBKLS0500, Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc, Orem, UT).

Xie J.Y., Chew L.A., Yang X., Wang Y., Qu C., Wang Y., Federici L.M., Fitz S.D., Ripsch M.S., Due M.R., Moutal A., Khanna M., White F.A., Vanderah T.W., Johnson P.L., Porreca F, & Khanna R. (2016). Sustained relief of ongoing experimental neuropathic pain by a CRMP2 peptide aptamer with low abuse potential. Pain, 157(9), 2124-2140.

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Western Blot Analysis of Tight Junction Proteins

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Tissue samples were prepared with inhibitors as described above. Samples were incubated for 10 min at 70°C and centrifuged for 5 min at 13,000 × g at 21°C to separate any solid particles before gel electrophoresis; 10 µg of total proteins from the tissue supernatant were loaded on TGX precast gels and separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Imobilon, Millipore) and blocked at room temperature for 1 h. Primary antibodies and dilutions are given above. All antibodies were diluted in 5% BSA in tris-buffered saline with Tween 20 (TBST). Immunoblots were revealed by enhanced chemiluminescence (WBKLS0500, Millipore) and imaged on photographic film. To visualize multiple bands on the same blot, blots were stripped with Restore Western Blot Stripping buffer (Pierce, Thermo Fisher). Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc.). Protein expression of TJ proteins were corrected for expression of the loading control α-tubulin to normalize for protein expression.

Cottier K.E., Galloway E.A., Calabrese E.C., Tome M.E., Liktor-Busa E., Kim J., Davis T.P., Vanderah T.W, & Largent-Milnes T.M. (2018). Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery. eNeuro, 5(4), ENEURO.0116-18.2018.

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Examining CRMP2 Phosphorylation in DRGs

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For examining the effect of (R)- or (S)-LCM on CRMP2 phosphorylation state, DRGs were treated for 30 min with the drugs and then lysed using RIPA buffer (50mM Tris-HCl, pH 7.4, 50mM NaCl, 2mM MgCl₂, 1% [vol/vol] NP40, 0.5% [mass/vol] sodium deoxycholate, and 0.1% [mass/vol] sodium dodecyl sulfate). Protease (Cat# B14002; Biotool) and phosphatase inhibitors (Cat# B15002, Biotool), and BitNuclease (Cat# B16002, Biotool) were included in RIPA buffer. Protein concentrations were determined using the BCA protein assay (Cat# PI23225, Thermo Fisher Scientific). Approximately 3µg of total proteins were loaded on an SDS-PAGE and then transferred to polyvinylidene difluoride membranes and blocked at room temperature for 1 hour in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1 % Tween 20), 5% non-fat dry milk. Primary antibodies used for probing were CRMP2 (Cat# C2993, Sigma), CRMP2 pSer522 (Cat# CP2191, ECM Biosciences), and βIII-tubulin (Cat# G712A, Promega,) diluted in TBST with 5% bovine serum albumin. Immunoblots were revealed by enhanced luminescence (WBKLS0500, Millipore) before exposure to a photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc).

Moutal A., Chew L.A., Yang X., Wang Y., Yeon S.K., Telemi E., Meroueh S., Park K.D., Shrinivasan R., Gilbraith K.B., Qu C., Xie J.Y., Patwardhan A., Vanderah T.W., Khanna M., Porreca F, & Khanna R. (2016). (S)-Lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology. Pain, 157(7), 1448-1463.

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