Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Representative western blot of stable HT29 line transduced with shSAE2 lentivirus showed lower SAE2, Oct-1 and ALDH1A1 protein levels than HT29 transduced with control non-silencing shRNA (shCtrl); GAPDH, loading control. Right panel: quantification of band intensity. ( b ) SAE2 knockdown decreased and SAE2 overexpression increased the occupancy of Oct-1 on the ALDH1A1 promoter as measured by ChIP assay. ( c ) SAE2 knockdown did not affect Oct-1 transcription as shown by real-time quantitative PCR measurement of mRNA of HT29 shCtrl and shSAE2 cells. ( d ) Representative western blot showing SAE2 knockdown reduced Oct-1 stability, as shown by treatment with 100 μg ml −1 CHX to block protein synthesis in HT29 shCtrl and shSAE2 cells; GAPDH, loading control. Quantification of band intensities (Oct-1 decay curve) in three independent experiments is shown below. ( e ) Representative western blot showing SAE2 knockdown-enhanced Oct-1 ubiquitination. HT29 shCtrl or shSAE2 cells were treated with or without proteasome inhibitor MG132 (10 μM) for 8 h, and IP was carried out using cell lysates, followed by western blot with an anti-Oct-1 or anti-ubiquitin antibody. NS, not significant; *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Western Blot, Transduction, shRNA, Over Expression, Real-time Polymerase Chain Reaction, Blocking Assay

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Mass spectrometry identified TRIM21 as an Oct-1-interacting protein. Representative silver stain of HCT116 cells transfected with Flag-tagged Oct-1 or empty vector (Ctrl) for 2 days, lysed and immunoprecipitated with anti-Flag antibody-conjugated M2 beads. Proteins identified in each group are indicated in the graphic. ( b ) Representative IP-western blot analysis of the interaction between endogenous TRIM21 and Oct-1 in HT29 cells. ( c ) Knockdown of TRIM21 delayed Oct-1 degradation. Representative western blot of Oct-1 level over time in HT29 cells transfected with control non-silencing siRNA (siCtrl) or siTRIM21 for 3 days, followed by 100 μg ml −1 CHX treatment; GAPDH, loading control. Oct-1 decay curve (right panel) was determined by quantifying three independent experiments. ( d ) TRIM21 knockdown resulted in increased Oct-1 protein level and decreased ubiquitination of Oct-1. Representative western blot of HT29 cells transfected with siCtrl and siTRIM21 treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and Oct-1, TRIM21 and ubiquitin were detected; GAPDH, loading control. ( e ) Overexpression of TRIM21 decreased Oct-1 and increased Oct-1 ubiquitination in cells. Representative western blot of HT29 cells were transfected with empty vector (Ctrl) or Flag-TRIM21 for 2 days, then treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and western blot was performed to detect Oct-1, TRIM21 and ubiquitin; GAPDH, loading control.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Mass Spectrometry, Silver Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Over Expression

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Representative IHC staining indicates higher TRIM21 level corresponding with lower Oct-1 in shSAE2 group tumour tissue compared with shCtrl group (scale bar, 100 μm). Tumour tissues were from LDA assay described in . ( b ) Western blot (left) and quantification of SAE2, Oct-1, TRIM21 and ALDH levels in the same tumour tissues as a ; GAPDH, loading control. ‘1, 2 and 3' indicate tumour tissues from different mouse. ( c ) TRIM21 promoter activity was inhibited by overexpression of SAE2 and Ubc9 but increased by SENP1 overexpression as determined by luciferase reporter assay. HT29 cells were transfected with empty vector (Ctrl) or SAE2, UBC9 or SENP1 expression plasmid together with TRIM21 promoter luciferase reporter and Renilla plasmids. Dual-luciferase activity was measured after 48 h and normalized results were analysed with two-tailed Student's t -test. ( d ) TRIM21 mRNA level was suppressed by SAE2 or Ubc9 overexpression and enhanced with SENP1 overexpression in HT29 cells as determined by quantitative PCR (qPCR). ( e ) IRF1 SUMOylation site mutant K78R induced higher TRIM21 mRNA level than wild-type (WT) IRF1 as determined by qPCR. ( f ) TRIM21 protein level was suppressed on SAE2 or Ubc9 overexpression but enhanced with SENP1 overexpression as indicated by western blot; GAPDH, loading control. ( g ) Western blot showed overexpression of K78R mutant-induced higher TRIM21 protein level than WT IRF1 in shCtrl HT29 cells; GAPDH, loading control. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Immunohistochemistry, Western Blot, Activity Assay, Over Expression, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. ( b ) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. ( c ) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH + cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. ( d ) Representative western blot of the samples from c ; quantification of the ALDH1A1 band intensity is shown on the bottom. ( e ) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). ( f ) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Over Expression, Stable Transfection, Generated, Expressing, Plasmid Preparation, Western Blot, FLAG-tag, Transfection, shRNA

Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Fibroblasts contribute to the up-regulation of OPN in HNC cells. (A) Immunohistochemical analysis of OPN protein expression in HNC tissues and matched adjacent normal tissues (Scale bar: 25 μm). Higher staining of OPN was observed in tumor cells of the edge of bulk tumors. (B) The mRNA and protein levels of OPN were determined in NFs, CAFs and cancer cells (derived from 4 HNC patients) using real-time PCR and western blotting. (C) OPN at the protein and mRNA level and in the supernatant was detected in 8 representative HNC cell lines and normal oral epithelial cells (titled normal) using western blotting, real-time PCR and ELISA. (D) The protein level, supernatant concentration and mRNA level of OPN were determined in CAL-27 and SCC-25 cells after co-culture with NFs. (E) The protein level, supernatant concentration and mRNA level of OPN were determined in NFs after co-culture with CAL-27 and SCC-25 cells. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: Immunohistochemical staining, Expressing, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay

Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Effects of stromal IL-6-induced OPN on promoting tumor growth and metastasis in vivo . (A) OPN overexpression in CAL-27 cells facilitated the xenograft tumor growth in nude mice. (B) Plasma OPN levels of the tumor-bearing mice from the normal group, CAL-27 group and CAL-27-OPN group. (C) Subcutaneous injection of OPN antibody (10 μg per tumor nodule) or IL-6 antibody (10 μg per tumor nodule) around the tumor partly inhibited NF-mediated tumor growth, and the combination of OPN antibody (5 μg per tumor nodule) and IL-6 antibody (5 μg per tumor nodule) exhibited a more powerful antitumor activity. (D) Plasma OPN levels of the tumor-bearing mice from the normal group, IgG treatment group, OPN antibody treatment group, IL-6 antibody treatment group and the combination group. (E) Overexpression of OPN in Rca-T cells promoted the formation and growth of metastatic nodules in nude mice (Scale bar, left: 5 mm; right: 100 μm). (F) Western blot analysis confirmed the exogenous expression of OPN in Rca-T cells, and plasma OPN levels of the mice involved in experimental metastasis were measured using ELISA. (G) Kaplan-Meier analyses of overall survival. (H, I and J) ROC curve analysis of the mRNA panel of OPN and IL-6 stratified by different groups in the validation set. ROC plots for the mRNA panel of OPN and IL-6 discriminated the five-year survival group from the death group (H), the TNM stage I group from the healthy controls (I), and the metastasis group from the non-metastasis group (J). AUC, area under the curve. (K) A proposed model illustrating the modulatory role of stromal IL-6-induced neoplastic OPN in controlling tumor growth and metastasis. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: In Vivo, Over Expression, Clinical Proteomics, Injection, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The bioactivity and targeting ability of KIT-PR1P. (A–D) The angiogenic ability of KIT-PR1P/VEGF and natural VEGF. N = 3. The Scale Bar = 200 μm ∗∗P < 0.01. (E) The targeting ability of KIT-PR1P/VEGF and PR1P/VEGF in hypoxic HK-2 cells was verified by immunofluorescence of the renal tubular injury marker Kim-1 and VEGF co-staining assay. VEGF (green), the renal tubular injury marker Kim-1 (red), DAPI (blue). Yellow staining indicates co-localization. The Scale Bar = 100 μm. (F) Fluorescence Integrated density of VEGF (35.34 ± 18.22, 17.48 ± 11.65, 4.89 ± 5.18) and Kim-1 (47.15 ± 23.47, 30.54 ± 15.99, 32.28 ± 17.85) in the KIT-PR1P/VEGF group, PR1P/VEGF group and PBS group. ∗∗∗∗P < 0.0001, ∗∗P < 0.01.All data are expressed as mean ± SD. (G) Fluorescence co-localisation analysis of Kim-1and VEGF in the KIT-PR1P/VEGF group. (H) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PR1P/VEGF group. (I) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PBS group.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Marker, Staining, Fluorescence

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The targeting ability of KIT-PR1P/VEGF in vivo . (A–B) Immunofluorescence co-staining assay. for VEGF (green) and the renal tubular injury marker Kim-1 (red) at 6 h and 24 h post-administration. DAPI (blue). The Scale Bar = 50 μm. (C) Fluorescence Integrated density of VEGF (24.60 ± 12.33, 10.79 ± 7.95, 4.47 ± 1.91, 3.17 ± 3.54) and Kim-1 (18.63 ± 9.67, 13.61 ± 6.10,13.23 ± 6.75, 13.85 ± 6.62) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001.All data are expressed as mean ± SD. (D) Fluorescence Integrated density of VEGF (42.82 ± 25.27, 7.13 ± 6.16, 8.94 ± 5.11, 3.61 ± 3.33) and Kim-1 (14.60 ± 7.62, 19.28 ± 10.51, 13.49 ± 6.20, 8.70 ± 6.06) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. ∗∗∗∗P < 0.0001. All data are expressed as mean ± SD. (E) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. (F) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. (G)Quantitative ELISA assay for VEGF in kidney at 6 h and 24 h post-injection. At 6 h post-injection, KIT-PR1P/VEGF = 0.616 ± 0.077 μg/g, KIT-PR1P = 0.429 ± 0.057 μg/g, PR1P/VEGF = 0.406 ± 0.033 μg/g, VEGF = 0.449 ± 0.027 μg/g, PBS = 0.266 ± 0.019 μg/g. At 24 h post-injection, KIT-PR1P/VEGF = 0.378 ± 0.041 μg/g, KIT-PR1P = 0.278 ± 0.031 μg/g, PR1P/VEGF = 0.254 ± 0.041 μg/g, VEGF = 0.185 ± 0.025 μg/g, PBS = 0.162 ± 0.060 μg/g. Data are presented as mean ± SD. N = 4, ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. (H) Quantitative ELISA assay for VEGF in serum. At 6 h post-injection, KIT-PR1P/VEGF = 3.100 ± 2.453 pg/ml, KIT-PR1P = 5.978 ± 0.813 pg/ml, PR1P/VEGF = 12.783 ± 2.710 pg/ml,VEGF = 7.455 ± 2.315 pg/ml, PBS = 8.765 ± 0.885 pg/ml. At 24 h post-injection, KIT-PR1P/VEGF = 8.042 ± 2.113 pg/ml, KIT-PR1P = 16.243 ± 5.550 pg/ml, PR1P/VEGF = 10.776 ± 4.668 pg/ml,VEGF = 13.899 ± 4.964 pg/ml, PBS = 16.059 ± 4.776 pg/ml. Data are presented as mean ± SD. N = 4, ∗P < 0.05. (I) Western blot of VEGF in KIT-PR1P/VEGF group, KIT-PR1P group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h and 24 h after injection, and Statistical analysis of Western blot of VEGF in KIT-PR1P/VEGF, KIT-PR1P, PR1P/VEGF, VEGF and PBS at I/R 6 h and 24 h after injection. (Mean ± SD, N = 4, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: In Vivo, Immunofluorescence, Staining, Marker, Fluorescence, Enzyme-linked Immunosorbent Assay, Injection, Western Blot

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The evaluation of renal function by serum Scr level after KIT-PR1P/VEGF intravenous injection. (A) Summary of Scr data for renal function after administration. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001. ∗∗P < 0.01. (B–D) Histogram of Scr assay for renal function after I/R injury and at 24 h, 72 h and 2w post-administration. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. N = 6.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Injection

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Morphology evaluation of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) Histopathological staining (H&E) for kidney pathological injury observation. Scale Bar = 20 μm. Red arrow indicated renal tubular injury; green asterisk indicated glomerulus. (D–F) Quantitative analysis of renal tubule injury based on H&E staining. (G–H) Masson staining of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (I–K) Quantitative analysis of the area of renal interstitial fibrosis in obstructed kidneys. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Injection, Staining

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Immunofluorescence staining of CD31 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Green fluorescence in this figure indicated areas stained positive for CD31, and blue fluorescence indicated DAPI. (A–C) Immunofluorescence staining for endothelial marker CD31 after I/R injury. Scale Bar = 20 μm. (D–F) Quantitative of Immunofluorescence staining for CD31. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Staining, Injection, Fluorescence, Marker

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: TUNEL staining and Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) TUNEL staining for apoptosis cells assessment (green). Scale Bar = 100 μm. (D–F) Quantitative of kidney TUNEL-positive cells. All data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (H–J) Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Red fluorescence indicated areas stained positive for cleaved-Caspase3, and blue fluorescence indicated DAPI. Immunofluorescence staining for cleaved-Caspase3 after I/R injury. Scale Bar = 50 μm. (K–M) Quantitative of Immunofluorescence staining for cleaved-Caspase3. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: TUNEL Assay, Staining, Immunofluorescence, Injection, Fluorescence

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Downstream pathway of KIT-PR1P/VEGF. (A) Immunofluorescence staining of p-VEGFR in ischemic kidney at 24 h intravenous injection of KIT-PR1P/VEGF after I/R injury and administration. Scale Bar = 20 μm. (B) Quantitative of IHF staining for p-VEGFR. All data are expressed as mean ± SD.∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (C) Western blotting assay of ischemia-reperfused kidneys for p-VEGFR, AKT, phospho-AKT, ERK, phospho-ERK at 24 h post administration. GAPDH was used as a control; (D) Statistical analysis of relative value for Western blot of p-VEGFR; (E) Statistical analysis of relative value for Western blot of AKT; (F) Statistical analysis of relative value for Western blot of p-AKT. (G) Statistical analysis of relative value for Western blot of ERK. (H) Statistical analysis of relative value for Western blot of p-ERK. N = 5. ∗∗∗∗p < 0 0.0001, ∗∗∗p < 0 0.001, ∗∗p < 0 0.01, ∗p < 0.05.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Staining, Injection, Western Blot, Control

Journal:

Article Title: Purification and Characterization of S -Adenosyl- l -Methionine: Desoxyhemigossypol-6- O- Methyltransferase from Cotton Plants. An Enzyme Capable of Methylating the Defense Terpenoids of Cotton 1

doi:

Figure Lengend Snippet: SDS-PAGE of the dHG-6-OMT active fraction from 2′,5′-ADP-Sepharose 4B affinity column (lane 1). Low-range silver-stain SDS-PAGE standards (Bio-Rad) (lane 2) are also shown.

Article Snippet: Ready gels (10%), the Silver-Stain Plus kit, and silver stain SDS-PAGE standard (low range) were purchased from Bio-Rad (Hercules, CA).

Techniques: SDS Page, Affinity Column, Silver Staining