HA-Tag antibody

Details for Product No. ABIN2443910, Supplier: Log in to see
Antigen
Reactivity
Tag
427
33
20
2
1
1
1
1
Host
Mouse
202
179
33
31
20
3
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
43
30
22
10
10
9
9
8
7
7
7
6
6
6
6
6
6
6
4
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
Application
Immunofluorescence (IF), Immunoprecipitation (IP), Immunohistochemistry (IHC), ELISA, Western Blotting (WB)
382
193
120
116
81
77
72
40
15
13
11
8
7
5
4
4
4
4
2
2
2
2
1
1
1
1
1
Options
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'Independent Validation' Badge
Antigen HA-Tag
Lot Number 16631508212-P
Method validated Western Blotting
Positive Control HA-fusion proteins expressed in NIH/3T3 cells
Notes Passed, regarding sensitivity and specificity. The HA-tag antibody ABIN2443910 does specifically recognize HA-tagged proteins in whole cell lysates from human tissue culture cells with no obvious background staining.
Primary Antibody ABIN2443910
Secondary Antibody Rabbit anti-Mouse IgG (whole molecule), HRP-linked (Sigma-Aldrich, A9044, lot number: 034M4761)
Protocol
  • Cultured NIH/3T3 cells heterologously expressing either a 26kDa (Fig. A) or 43kDa (Fig. B) HA-tag-fusion protein were rinsed with PBS and lysed in 6M Urea/20mM Tris.
  • 40µg total protein were used for Western blot analysis.
  • Proteins were denatured and separated on 10% SDS-PAGE (Laemmli 1970) and blotted to Amersham Protran Premium 0.2 NC (GE Healthcare, 10600004, lot A10043108) (Towbin et al., 1979).
  • Blocking of membrane in 5% skim milk in TBST (50 mM Tris-HCl, pH 7.4, 150mM NaCl, 0.1% Tween 20) for 30min at RT.
  • Incubation with primary antibody ABIN2443910 diluted 1:600 in 5% skim milk in TBST overnight at 4°C.
  • Washing in TBST for 30min at RT.
  • Incubation with secondary antibody secondary antibody: rabbit-anti-mouse IgG (whole molecule), HRP-linked (Sigma-Aldrich, A9044, lot 034M4761) diluted 1:10000 in 5% skim milk in TBST for 45min at RT.
  • Washing in TBST for 30-45min at RT.
  • Chemiluminescence detection using Clarity Western ECL Substrate (BioRad, 170-5061) according to the supplier’s recommendations and image capture via X-ray films.
Experimental Notes A dilution factor <1000 is recommended for Western blot on cellular lysates with ABIN2443910.
Validation Images
Western Blotting validation image for anti-HA-Tag antibody (ABIN2443910) HA-tagged fusion proteins were expressed in NIH/3T3 cells. Whole cell lysates were se...
'Independent Validation' Badge
Antigen HA-Tag
Lot Number 16631508212-P
Method validated Immunocytochemistry
Positive Control HA-fusion proteins transiently expressed in NIH/3T3 cells
Notes Passed, regarding sensitivity and specificity. The antibody specifically detects the HA-fusion protein in immuno-cytochemical experiments. No obvious background staining was found.
Primary Antibody ABIN2443910
Secondary Antibody MFP488 goat anti-mouse IgG (H+L) (MoBiTec, MFP-A1029, lot 2803101)
Protocol
  • NIH/3T3 cells (ATCC) were grown on cover slips in DMEM, 10% fetal bovine serum, 5% penicillin/streptomycin (all Gibco) at 37°C in 5% CO2.
  • Cells were transfected using Metafectene Pro (Biontex, T040-1.0, lot AD1.13) according to the manual with a plasmid encoding an HA-tag fusion protein that is expected to localize to the nuclear membrane and the cytoplasm.
  • 24h post-transfection cells were fixed in 3.7% paraformaldehyde in PBS for 15min and processed for immuno-cytology.
  • Unspecific binding sites were blocked in PBST (phosphate buffered saline containing 0.15% bovine serum albumin, 0.1% Tween-20) for 1h.
  • Cells were incubated with the primary antibody ABIN2443910 diluted 1:100 in PBS at 4°C overnight.
  • Cells were washed in TBST (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.1% Tween 20) for 15min.
  • Incubation with the secondary antibody MFP488 goat anti-mouse IgG (H+L) (MoBiTec, MFP-A1029, lot 2803101) diluted 1:600 in PBS and DAPI (stock solution 10µg/µl, diluted 1:500) for 1h at 37°C.
  • Cells were washed in TBST for 15min.
  • Cells were embedded in Fluorescent Mounting Medium (Dako, S3023, lot 10090890).
  • Images were taken by confocal microscopy (LSM 510, Zeiss), and processed using Adobe Photoshop 5.0.
Experimental Notes none
Validation Images
Immunocytochemistry validation image for anti-HA-Tag antibody (ABIN2443910) HA-tagged fusion proteins were expressed in NIH/3T3 cells. The protein of interest is...
Sequence YPYDVPDYA
Clone HA-C5
Isotype IgG3
Purification Purified
Alternative Name HA Tag
Target Type Tag
Application Notes Optimal working dilution should be determined by the investigator.
Restrictions For Research Use only
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