V5 tag antibody [SV5-Pk1] (ab27671)

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Anonymous Abreview submitted on 29 July 2009
Application:	Immunocytochemistry
V5 tag antibody [SV5-Pk1] (ab27671)
Sample	Chinese Hamster Cultured Cells (CHO cells overexpressing a V5-tagged protein)
Specification	CHO cells overexpressing a V5-tagged protein
Fixative:	Paraformaldehyde
Permeabilization:	Yes - 0.1% Triton X-100 in PBS
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 3 % · Temperature: 22°C
Other product details
Dilution	1/1000
Incubation time:	12 hours  Temperature: 4°C Diluent: 3% BSA + 2% Goat Serum in PBS
Secondary antibody
Name:	Non-Abcam antibody was used: Alexa Fluor® 488 goat anti-mouse IgG (H+L) Conjugation: Alexa Fluor® 488
Dilution:	1/1000
Additional data
Rating	Excellent
Additional notes	As shown in the image below (Blue:DAPI Green: V5-tag), this V5-tag antibody had a very good staining in the CHO cells overexpressing a V5-tagged protein. However, this antibody did have a little background staining in the negative control cells (not shown here). Therefore, a negative control is recommended in your experiment.
Image	V5 tag antibody [SV5-Pk1] for Immunocytochemistry (Chinese Hamster) Copyright image used under license

Anonymous Abreview submitted on 14 May 2009
Application:	Western blot
V5 tag antibody [SV5-Pk1] (ab27671)
Sample	Human Cell lysate - whole cell (HEK293)
Loading amount:	5 µg
Specification	HEK293
Gel Running Conditions:	Reduced Denaturing (10)
Blocking step:	Milk as blocking agent for  1 hour  · Concentration: 5 % · Temperature: 22°C
Other product details
Dilution	1/2500
Incubation time:	12 hours  Temperature: 4°C
Secondary antibody
Name:	Non-Abcam antibody was used: PEROXIDASE LABELED Anti-mouse IgG (H+L) Conjugation: HRP
Dilution:	1/5000
Detection
Detection method:	ECL
Exposure:	5 minutes and 0 seconds
Bands:	Specific: ~100 kDa
Additional data
Rating	Excellent
Additional notes	Lane 1&2 are HEK293 cell lysates expressing V5 tagged protein (Lane 2 are from longer incubation time after transfection). Lane 3 is native HEK293 cell lysate. This V5 tag antibody is highly specific in my experimental conditions.
Image	V5 tag antibody [SV5-Pk1] for Western blot (Human) Copyright image used under license

review by Craig Faulkner submitted on 16 May 2008
Application:	Western blot
V5 tag antibody [SV5-Pk1] (ab27671)
Sample	Fruit fly (Drosophila melanogaster) Cell lysate - whole cell (Transfected S2 Cells expressing V5 Tagged protein)
Loading amount:	20 µg
Specification	Transfected S2 Cells expressing V5 Tagged protein
Treatment	1mM CuSO4 for 24hrs
Gel Running Conditions:	Reduced Denaturing (10%)
Blocking step:	Milk as blocking agent for  2 hours  · Concentration: 5 %
Other product details
Dilution	1/5000
Incubation time:	16 hours
Secondary antibody
Name:	Non-Abcam antibody was used: Goat anti-mouse AP Conjugation: Alkaline Phosphatase
Dilution:	1/1000
Detection
Detection method:	AP
Exposure:	5 minutes and 0 seconds
Bands:	Specific: ~37 kDa   Non-specific: ~42 kDa
Positive control:	Positive control was S2 cells transfected with pMT p47phox and was a very intense single band at 47kDa with little to no background.
Negative control:	Negative control was non-transfected S2 cells. No staining observed
Additional data
Rating	Excellent
Additional notes	The protein of interest was V5 tagged C5a Receptor Like 2 (C5aRL2). Would recommend this antibody over the polyclonal V5 antibody ab9116 for western blot applications
Image	V5 tag antibody [SV5-Pk1] for Western blot (Fruit fly (Drosophila melanogaster)) Copyright image used under license