6X His tag® antibody - ChIP Grade (ab9108)

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review by Kang sik seo submitted on 15 January 2010
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Cell lysate - whole cell (HeLa)
Loading amount:	20 µg
Specification	HeLa
Gel Running Conditions:	Reduced Denaturing (10%)
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 5 % · Temperature: 25°C
Primary Antibody
Dilution	1/1000
Incubation time:	12 hours  Temperature: 4°C
Secondary Antibody
Name:	Non-Abcam Antibody was used: Anti-Rabbit IgG HRP Conjugation: HRP
Detection
Detection method:	ECL
Exposure:	3 minutes and 0 seconds
Bands:	Specific: 120 kDa
Additional Data
Rating	Average
Additional notes	Cell lysed in RIPA buffer supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche #11 836 153 001) PVDF membrane Lane 1: Vehicle Lane 2: Overexpression of His-HIF-1alpha
Image	6X His tag® antibody - ChIP Grade for WB (Human) Copyright image used under license

review by Chien Hsin Lee submitted on 3 September 2009
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Recombinant protein (TNT system, in vitro translation)
Loading amount:	2 µg
Specification	TNT system, in vitro translation
Gel Running Conditions:	Non-reduced Denaturing (10%)
Blocking step:	Milk as blocking agent for  16 hours  · Concentration: 5 % · Temperature: 4°C
Primary Antibody
Dilution	1/1000
Incubation time:	1 hour  Temperature: 22°C Diluent: 5% Milk
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti-Rabbit, HRP conjugate Conjugation: HRP
Dilution:	1/10000
Detection
Detection method:	ECL
Exposure:	1 minute and 0 seconds
Bands:	Specific: 60-70 kDa
Additional Data
Rating	Good
Image	6X His tag® antibody - ChIP Grade for WB (Human) Copyright image used under license

Anonymous Abreview submitted on 1 December 2008
Application:	Immunocytochemistry/ Immunofluorescence
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Rat Cell (Rat2 Cells)
Specification	Rat2 Cells
Fixative:	Paraformaldehyde
Permeabilization:	Yes - 0.1% Saponin
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 0.1 % · Temperature: 37°C
Primary Antibody
Dilution	1/500
Incubation time:	1 hour  Temperature: 37°C Diluent: PBS + 0.1% BSA
Secondary Antibody
Name:	Non-Abcam Antibody was used: Alexa 555 donkey anti rabbitt Conjugation: Alexa Fluor® 555
Dilution:	1/1000
Additional Data
Rating	Good
Additional notes	His-tagged construct was transfected into Rat2 cells. Cells were stained with His antibody and those that are transfected appear in red.
Image	6X His tag® antibody - ChIP Grade for ICC/IF (Rat) Copyright image used under license

Anonymous Abreview submitted on 14 October 2008
Application:	Immunoprecipitation
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Rat Cell lysate - whole cell (Cos7 cells)
Total protein in input:	300 µg
Specification	Cos7 cells
Immuno- precipitation step	Protein G
Primary Antibody
Dilution	1/100
Incubation time:	2 hours  Temperature: 4°C Diluent: 5% milk in pbs with 0.5% tween
Western Blot Antibody
Name:	Non-Abcam Antibody was used: Dynein light intermediate chain 1 Conjugation: HiLyte Plus™ 647
Dilution:	1/1500
Additional Data
Rating	Good
Additional notes	Although there is a lot of background from my primary antibody, the protein of interest (LIC1) is at 57kD. This protein is pulled down by the his-tagged construct that was transfected into cos7 cells. The first lane is the ladder (in white) the next three lanes are without his antibody (input, sup, pellet), and there is no LIC that sticks to the beads without antibody. The next three lanes are with his antibody (input, sup, pellet), and the LIC protein is IPed, as seen in the pellet. I used a secondary antibody, the primary was not conjugated
Image	6X His tag® antibody - ChIP Grade for IP (Rat) Copyright image used under license

review by Vladimir Milenkovic submitted on 3 June 2008
Application:	Immunoprecipitation
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Cell lysate - whole cell (HEK 293 cells transfected with Best1-His)
Total protein in input:	25 µg
Specification	HEK 293 cells transfected with Best1-His
Immuno- precipitation step	Protein G
Primary Antibody
Incubation time:	16 hours  Temperature: 4°C Diluent: 1%TX-100, 10% glycerol, 1XPBS
Western Blot Antibody
Name:	Abcam antibody: Bestrophin antibody
Dilution:	1/10000
Additional Data
Rating	Good
Additional notes	Line 1: Non transfected HEK 293 cells Line 2: Lysate of HEK 293 cells expressing Best1-His Line 3: IP with anti His Ab
Image	6X His tag® antibody - ChIP Grade for IP (Human) Copyright image used under license

review by Vladimir Milenkovic submitted on 3 June 2008
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Cell lysate - whole cell (HEK 293 cells transfected with CACNB3-His)
Loading amount:	25 µg
Specification	HEK 293 cells transfected with CACNB3-His
Gel Running Conditions:	Reduced Denaturing
Blocking step:	Milk as blocking agent for  30 mins  · Concentration: 5 % · Temperature: 25°C
Primary Antibody
Dilution	1/2000
Incubation time:	16 hours  Temperature: 4°C Diluent: 1X PBS/Tween, 5% milk
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti rabbit HRP Conjugation: HRP
Dilution:	1/750
Detection
Detection method:	ECL+
Exposure:	5 minutes and 0 seconds
Bands:	Specific: 57 kDa
Additional Data
Rating	Good
Additional notes	Line 1: Non transfected HEK 293 cells Line 2: HEK 293 cells transfected with CACNB3-His
Image	6X His tag® antibody - ChIP Grade for WB (Human) Copyright image used under license

review by Vladimir Milenkovic submitted on 5 June 2008
Application:	Immunocytochemistry/ Immunofluorescence
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Cell (CACNB3 with His tag expressed in HEK293 cells)
Specification	CACNB3 with His tag expressed in HEK293 cells
Fixative:	Paraformaldehyde
Permeabilization:	Yes - 0.5% TX100
Blocking step:	Serum as blocking agent for  20 mins  · Concentration: 5 % · Temperature: 25°C
Primary Antibody
Dilution	1/500
Incubation time:	16 hours  Temperature: 4°C Diluent: 1% goat serum, 0.1%TX100, 1XPBS
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti rabbit Alexa Fluor 488 Conjugation: Alexa Fluor® 488
Dilution:	1/750
Additional Data
Rating	Excellent
Additional notes	HEK 293 cells were transfected wirh CACNB3-His and stained with anti His Ab 24 h after transfection.
Image	6X His tag® antibody - ChIP Grade for ICC/IF (Human) Copyright image used under license

Anonymous Abreview submitted on 29 November 2007
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Recombinant protein (produced in insect cells)
Loading amount:	5 µg
Specification	produced in insect cells
Treatment	cobalt affinity purification, FPLC
Gel Running Conditions:	Reduced Denaturing
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 5 % · Temperature: 23°C
Primary Antibody
Dilution	1/1000
Incubation time:	1 hour  Temperature: 23°C Diluent: PBST
Secondary Antibody
Name:	Non-Abcam Antibody was used: goat anti-rabbit HRP Conjugation: HRP
Dilution:	1/2000
Detection
Detection method:	ECL
Exposure:	30 minutes and 0 seconds
Bands:	Specific: 32 kDa
Additional Data
Rating	Excellent
Additional notes	varying fractions of an FPLC column purification
Image	6X His tag® antibody - ChIP Grade for WB (Human) Copyright image used under license

Anonymous Abreview submitted on 22 November 2007
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Saccharomyces cerevisiae Cell lysate - whole cell (Strain BY4741)
Loading amount:	100000 cells
Specification	Strain BY4741
Blocking step:	BSA as blocking agent for  2 hours  · Concentration: 5 % · Temperature: 25°C
Primary Antibody
Dilution	1/1000
Incubation time:	8 hours  Temperature: 4°C Diluent: TBS-T
Secondary Antibody
Name:	Non-Abcam Antibody was used: ImmunoPure Antibody from Pierce Conjugation: HRP
Dilution:	1/30000
Detection
Detection method:	ECL
Exposure:	15 seconds
Bands:	Specific: 36 kDa   Non-specific: 30 kDa
Positive Control:	Strain expressing a His-tagged version of Spp1
Negative Control:	Strain expressing an untagged version of Spp1
Additional Data
Rating	Excellent
Additional notes	The lower band mind also be specific, representing a degradation product of His-Spp1.
Image	6X His tag® antibody - ChIP Grade for WB (Saccharomyces cerevisiae) Copyright image used under license

Anonymous (click to contact this user) Abreview submitted on 24 January 2007
Application:	ELISA
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Mouse Recombinant protein (Interaction between 2 extracellular matrix protein)
Specification	Interaction between 2 extracellular matrix protein
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 1 mg/ml
Type:	Indirect
Primary Antibody
Dilution	1/500
Incubation time:	1 hour
Secondary Antibody
Name:	Non-Abcam Antibody was used: Swine anti-rabbit Conjugation: Alkaline Phosphatase
Dilution:	1/500
Additional Data
Rating	Good
Additional notes	Buffer used for washing and incubation was PBS with 0.5 mg/ml BSA and 0,2% Tween-20

Anonymous (click to contact this user) Abreview submitted on 22 January 2007
Application:	Flow Cytometry
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Saccharomyces cerevisiae Cell (surface-displayed construct on yeast cell)
Specification	surface-displayed construct on yeast cell
Preparation:	Cell harvesting/tissue preparation method: Sample buffer:
Fixation:
Permeabilization:	No
Gating Strategy:
Primary Antibody
Dilution	1/100
Incubation time:	30 minutes
Secondary Antibody
Name:	None used
Additional Data
Rating	Excellent
Additional notes	This antibody was labeled with Alexa 647 and used to sort populations of cells with his tag present.The image shows FACS data of a population of cells, some of which express a his tagged construct (right peak), some without (left peak).
Image	6X His tag® antibody - ChIP Grade for Flow Cyt (Saccharomyces cerevisiae) Copyright image used under license

review by N Chapman submitted on 19 April 2006
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Recombinant protein (Recombinant His-tagged human RelA NF-kappaB N-term)
Loading amount:	10 µg
Specification	Recombinant His-tagged human RelA NF-kappaB N-term
Blocking step:	Milk as blocking agent for  1 hour  · Concentration: 10%
Primary Antibody
Dilution	1/1000
Incubation time:	3 hours
Secondary Antibody
Name:	Non-Abcam Antibody was used: Polyclonal goat anti-rabbit immunoglobulin HRP Conjugation: HRP
Concentration:	300 µg/ml
Detection
Detection method:	ECL+
Exposure:	1 minute and 0 seconds
Bands:	Specific: 37 kDa   Non-specific: 12, >200 kDa
Positive Control:	This sample
Negative Control:	Non-transformed E.coli lysates and transformed, non-induced E.coli lysates
Additional Data
Rating	Excellent
Additional notes	Very clean antibody; clear image of recombinant N-terminal domain of RelA when expressed in E.coli. Buffer was standard PBS + 0.05% (v/v) tween-20. Block was this buffer + 10% (w/v) low-fat dried milk (marvel). Seconadry, listed below used at 1:20,000 dilution (Polyclonal goat anti-rabbit immunoglobulin HRP; DakoCytomation p0448). Figure legend: A) Blot 1 minute; B) Blot 15 minutes: C- control, non-transformed lysate; C+I control + 1mM IPTG; T- E.coli Lysate harbouring His-tag-RelA NTD, non-induced; T+I Induction of His-tag RelA NTD (two lanes) 1 mM IPTG.
Image	6X His tag® antibody - ChIP Grade for WB (Human) Copyright image used under license

Anonymous (click to contact this user) Abreview submitted on 14 September 2005
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Sample	Human Cell lysate - whole cell (293-EBNA-HAD1)
Specification	293-EBNA-HAD1
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 5%
Primary Antibody
Dilution	1/2500
Incubation time:	16 hours
Secondary Antibody
Name:	Abcam antibody: Rabbit IgG secondary antibody - H&L
Dilution:	1/5000
Detection
Detection method:	ECL
Exposure:	5 minutes and 0 seconds
Bands:	Specific: 64 kDa   Non-specific: 40, 55, 60 kDa
Negative Control:	293-EBNA
Additional Data
Rating	Excellent

review by Anna Castano submitted on 19 April 2004
Application:	ELISA
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	The 6XHis antibody is better than many of the antibodies that we have been using. It's nice to be able to use smaller amounts of antibody to detect our proteins.
Dilution notes	ELISA 1:1000-1:5000 FACS 1:1000-1:4000 IFA 1:1000-1:5000 Western 1:1000-1:6000

review by Alex Georgiou submitted on 18 March 2004
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	Good and clear signals on the membrane, however there are still some other bands (non-specific). Happy with the results. Will order again.
Dilution notes	1:2500 in 1% BSA 1:5000 in 1% BSA
Abcam response	Thank you for your valuable feedback information about this product. Ab9108 is a polyclonal antibody, so it recognizes different epitopes. At high concentration multiple bands are often seen. We would suggest decreasing the antibody concentration and/or the incubation period.

review by Megan Lloyd submitted on 11 March 2004
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	This antibody was used at 1/1000 in TBS-T to detect expression of recombinant his-tagged proteins in E.coli BL21. It worked very well with very low background. The detection system used was the ECL Plus Western blotting kit. I recommend it.
Dilution notes	1/1000

review by Didier Petiteville submitted on 4 December 2003
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	ab9108 was successfully used for immunoblot detection of purified recombinant proteins expressed in insect cells through the expression vector pFastBac HT (Invitrogen). Antibody recognized well proteins in total cell lysate. Dilution used (1/2500) Detection: HRP/ECL Worked very well, no background or unspecific bands
Dilution notes	Dilution used (1/2500)

review by Mark Segal submitted on 5 June 2003
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	The antibody has worked well to detect His tagged proteins in an immunoblot.
Dilution notes	We use 1:1000 dilution in TBS with BSA

review by Vincent Smith submitted on 7 April 2003
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	This antibody worked very well when used in western blots to detect 6xHis tagged proteins expressed in E.Coli.
Dilution notes	- Recommended dilutions: 1:1000 with goat anti-rabbit AP conjugate (Sigma A3937) secondary at 1:5000. Development with Promega Western blue stabilised AP substrate. The antibody was diluted in PBS, 0.1% Tween 20 without carrier protein.

review by Wendy Gion /EST Biology submitted on 12 August 2002
Application:	Western blot
6X His tag® antibody - ChIP Grade (ab9108)
Additional Data
Notes	I used the rabbit anti-6His antibody on a Western blot to look for the production of a His-tagged fusion protein produced in E. Coli. I used a 1:5000 dilution of the anti-6His antibody and a 1:5000 dilution of the secondary antibody (conjugated to HRP). The ECL kit (Amersham) was used for detection. Western blot was successful, with only two background bands visible besides the band of interest.
Dilution notes	1:5000 .