MKRN3 抗体 (AA 160-189)
(1 validation)-
- 抗原 See all MKRN3 products
- MKRN3 (Makorin Ring Finger Protein 3 (MKRN3))
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抗原表位
- AA 160-189
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适用
- 人
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宿主
- 兔
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克隆类型
- 多克隆
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标记
- This MKRN3 antibody is un-conjugated
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应用范围
- Western Blotting (WB)
- 纯化方法
- This antibody is purified through a protein A column, followed by peptide affinity purification.
- 免疫原
- This MKRN3 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 160-189 amino acids from the Central region of human MKRN3.
- 克隆位点
- RB27251
- 亚型
- IgG
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anti-Makorin Ring Finger Protein 3 (MKRN3) (AA 437-507) antibody
MKRN3 适用: 人 ELISA, IF 宿主: 小鼠 Monoclonal 2E10 unconjugated
anti-Makorin Ring Finger Protein 3 (MKRN3) (AA 1-260) antibodyMKRN3 适用: 人 WB 宿主: 兔 Polyclonal unconjugated
anti-Makorin Ring Finger Protein 3 (MKRN3) antibodyMKRN3 适用: 人 WB, ELISA, IHC 宿主: 兔 Polyclonal unconjugated
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- 应用备注
- WB: 1:1000
- 限制
- 仅限研究用
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- by
- Department of Medical Biochemistry and Microbiology, Uppsala University
- No.
- #101148
- 日期
- 2017.08.10
- 抗原
- MKRN3
- Lot Number
- SA100913BT
- Method validated
- Western Blotting
- Positive Control
H1299 cells transfected with HA-MKRN3 expression vector; the full-length MKRN3 CDS has been inserted downstream of an HA-tag in plasmid pcDNA3.1
- Negative Control
H1299 cells transfected with HA-MKRN1 or HA-MKRN2 expression plasmid; the full-length MKRN1 or MKRN2 CDS has been inserted downstream of an HA-tag in plasmid pcDNA3
Empty plasmid pcDNA3.1
- Notes
Passed. ABIN2840417 specifically recognizes MKRN3 ectopically expressed in H1299 cells lysates.
- Primary Antibody
- ABIN2840417
- Secondary Antibody
- IRDye 680LT donkey anti-rabbit IgG (H+L) antibody (LI-COR, 925-68023)
- Full Protocol
- Grow H1299 cells (human lung carcinoma cell line) in DMEM medium (ThermoFischer Scientific, 41965-039) supplemented with 10% fetal bovine serum (ThermoFischer Scientific, 10500064) and antibiotics (1X Penicillin-Streptomycin mix, 15140122, ThermoFischer Scientific), at 37°C and 7% CO2 in 12-well plates.
- Transfect H1299 cells with plasmids expressing either HA-MKRN1, HA-MKRN2, HA-MKRN3 or with an empty plasmid pcDNA3.1 (ThermoFischer Scientific, V79020) with max 2µg of plasmid using Turbofect transfection reagent (ThermoFisher Scientific, R0531) following the manufacturer´s instructions.
- Lyse cells in volume RIPA buffer.
- Determine total protein content of the lysates using a Bradford protein assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, 5000006).
- Denature 80µg of total protein for 5min at 95°C in 20µl of 2x Laemmli SDS sample buffer and subsequently separate them on a denaturing Any kD Mini-PROTEAN TGX precast protein gel (Bio-Rad, 4569033) in Mini-PROTEAN Electrophoresis Cells for 1h at 180V.
- Transfer proteins onto Amersham Protran 0.45µm nitrocellulose filter (GE Healthcare Life Sciences) with transfer buffer (25mM Tris, 192mM glycine, 20% methanol) in a Western blotting system for at 4°C for 3h at 120mA.
- Block the membrane with Odyssey Blocking Buffer (LI-COR, 927-50100) for 15min at RT.
- Incubation with primary
- rabbit anti-MKRN3 antibody (antibodies-online, ABIN2840417, lot SA100913BT) diluted 1:1000 in Odyssey Blocking Buffer ON at 4°C or
- mouse anti-HA-tag antibody (BioLegend, 901501) diluted 1:2000 in Odyssey Blocking Buffer ON at 4°C.
- Wash membrane 5x for time with PBS supplemented with 0.1% Tween 20.
- Incubation with secondary
- IRDye 680LT donkey anti-rabbit IgG (H+L) antibody (LI-COR, 925-68023) or
- IRDye 800CW donkey anti-mouse IgG (H+L) antibody (LI-COR, 926-32212)
- diluted 1:10000 in Odyssey Blocking Buffer for 30 min at RT.
- Wash membrane 5x for time with PBS supplemented with 0.1% Tween 20.
- Scan the membrane with LI-COR Odyssey CLX Scanner.
- Experimental Notes
ABIN2840417 is specific towards the expressed HA-MKRN3: it does not recognize HA-MKRN1 (which has 48% identity with MKRN3) and HA-MKRN2 (has 37% identity with MKRN3) ectopically expressed in H1299 cells.
An extraneous band can be detected with the antibody around 40kDa. This band is recognized with both anti HA- and anti MKRN3-antibodies. This suggests either C-terminal degradation product of HA-MKRN3 or that HA-MKRN3 mRNA has been not efficiently translated.
A smear is visible for HA-MKRN3 protein with both antibodies, which may indicate that the protein undergoes self-ubiquitination or forms higher order structures.
At present we have had difficulties to use the antibody to detect endogenous MKRN3 protein. This is probably due to the fact that endogenous MKRN3 protein levels can be very low is some cell lines.
生效 #101148 (Western Blotting)!['独立验证'标志]( "成功验证")
!['独立验证'标志]( "成功验证")
Validation Images![A. Western blot of cell lysates of H1299 human lung carcinoma cells transfected with an empty, HA-tagged MKRN1, MKRN2, or MKRN3 expression plasmids (from left) using primary antibody ABIN2840417. B. Western blot analysis of the same lysates using an HA-tag specific antibody.]()
A. Western blot of cell lysates of H1299 human lung carcinoma cells transfected with an empty, HA-tagged MKRN1, MKRN2, or MKRN3 expression plasmids (from left) using primary antibody ABIN2840417. B. Western blot analysis of the same lysates using an HA-tag specific antibody.
Full Methods -
- 状态
- Liquid
- 缓冲液
- Purified polyclonal antibody supplied in PBS with 0.09 % (W/V) sodium azide.
- 储存液
- Sodium azide
- 注意事项
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
- 储存条件
- 4 °C,-20 °C
- 储存方法
- Maintain refrigerated at 2-8 °C for up to 6 months. For long term storage store at -20 °C in small aliquots to prevent freeze-thaw cycles.
- 有效期
- 6 months
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- 抗原
- MKRN3 (Makorin Ring Finger Protein 3 (MKRN3))
- 别名
- MKRN3 (MKRN3 产品)
- 别名
- CPPB2 antibody, D15S9 antibody, RNF63 antibody, ZFP127 antibody, ZNF127 antibody, D7H15S9-1 antibody, Zfp127 antibody, makorin ring finger protein 3 antibody, makorin, ring finger protein, 3 antibody, MKRN3 antibody, Mkrn3 antibody
- 背景
- The protein encoded by this gene contains a RING (C3HC4) zinc finger motif and several C3H zinc finger motifs.
- 分子量
- 55645
- 基因ID
- 7681
- NCBI登录号
- NP_005655
- UniProt
- Q13064
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