Anti-6X His tag® antibody

• ICC/IF

AbReview10865****

Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody (AB9108)

ab9108 staining CACNB3 with His tag expressed in HEK293 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 5% serum for 20 minutes at 25°C. Samples were incubated with primary antibody (1/500 in 1% goat serum, 0.1% TX100 and PBS) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody (1/750).

This image is courtesy of Vladimir Milenkovic

• ChIP

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ChIP - Anti-6X His tag® antibody (AB9108)

A stably transfected 293T human cell line harbouring the GAL4 upstream activation sequence was transiently transfected with a Myc or His - tagged GAL4 DNA Binding Domain construct. 48 hours post transfection Chromatin was prepared according to the Abcam X-ChIP protocol. The ChIP was performed with 25 ug chromatin, 5ug of antibody and 20 ul of Protein A/G beads. A non-specific antibody was used as the negative control. The immunoprecipitated DNA was quantified by real time PCR (SYBR Green approach).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody (AB9108)

Immunofluorescent analysis of 4% paraformaldehyde (PFA) -fixed, permeabilized with 0.1% Triton X-100 in CHO cells transfected with 12 tags constructs (CHO-12 tags) labelling 6xHIS with ab9108 at 1/1000 dilution (1µg/mL), followed by Donkey Anti-rabbit IgG (H&L), Alexa Fluor® 594 antibody at 1/500 dilution (4 µg/mL) (Red). Nucleus was counterstained with DAPI (Blue). Bottom panel images showed Parallel staining in parental CHO cell line.

• WB

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Western blot - Anti-6X His tag® antibody (AB9108)

Use of ab9108 in western blotting at 1 : 1000 against Epitope Tag Positive Control lysate (ab4524).
Lane 1 : 2 µl lysate
Lane 2 : 1 µl lysate
Lane 3 : 0.5 µl lysate
Lane 4 : 0.25 µl lysate Use of ab9108 in western blotting at 1 : 1000 against E.coli Positive Control (Escherichia coli ) Whole Cell Lysate - expressing 11 different epitope tag (ab5395). Lane 1 : 2 µl lysate Lane 2 : 1 µl lysate Lane 3 : 0.5 µl lysate Lane 4 : 0.25 µl lysate

All lanes:

Western blot - Anti-6X His tag® antibody (ab9108)

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• WB

AbReview41922****

Western blot - Anti-6X His tag® antibody (AB9108)

All lanes:

Western blot - Anti-6X His tag® antibody (ab9108) at 1/4000 dilution

Lane 1:

HeLa whole cell lysate - transfected with empty vector at 10 µg

Lane 2:

HeLa whole cell lysate - transfected with His-tagged gene at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG polyclonal at 1/5000 dilution

Observed band size: 35 kDa

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Exposure time: 30s

This image is courtesy of an anonymous Abreview

• WB

Supplier Data

Western blot - Anti-6X His tag® antibody (AB9108)

Lane 1 and 4 : Molecular weight Standards. Primary and secondary antibody incubation at RT for 1 hour.

All lanes:

Western blot - Anti-6X His tag® antibody (ab9108) at 1 µg/mL

Lane 1:

Purified 12-Tag at 2 µg

Lane 2:

Purified 12-Tag at 0.2 µg

Lane 3:

The cell lysates of CHO-12 Tags at 1 µg

Lane 4:

The cell lysates of CHO at 2 µg

Secondary

All lanes:

Goat anti-rabbit, HRP conjugated at 0.2 µg/mL

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• WB

Supplier Data

Western blot - Anti-6X His tag® antibody (AB9108)

Lanes 1 - 5 :  Green – ab282749 at 1 in 500 dilution, observed from 120 – 180 kDa
Lanes 6 - 8 :  Red – Rabbit polyclonal to 6X His tag^® (ab9108) at 1 in 1000 dilution, observed from 120 – 160 kDa

Lanes 9 & 10 : Black – Goat polyclonal to DDDDK tag (Binds to FLAG^® tag sequence) at 1 μg/mL, observed at 180 kDa

ab282749 was shown to bind specifically to SARS-CoV and SARS-CoV-2 Spike Glycoproteins in Western blot. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.

Blocking buffer : 3% milk in TBS-0.1% Tween^® 20 (TBS-T).

Lanes 1 - 5:

Western blot - Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody [8B12-C2] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/sars-cov-2-spike-glycoprotein-s1-antibody-8b12-c2-bsa-and-azide-free-ab282749'>ab282749</a>) at 1/500 dilution

Lanes 6 - 8:

Western blot - Anti-6X His tag® antibody (ab9108) at 1/1000 dilution

Lanes 9 - 10:

Goat polyclonal to DDDDK tag at 1 µg/mL

Lanes 1 and 6:

Western blot - Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-coronavirus-sars-cov-2-spike-glycoprotein-s1-active-ab273068'>ab273068</a>) at 0.5 µg

Lanes 2 and 7:

SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein at 0.2 µg

Lanes 3 and 8:

SARS-CoV Spike S1+S2 ECD-His Recombinant Protein at 0.2 µg

Lanes 4 and 9:

SARS-CoV-2 3xFlag Spike Protein Transfected Expi whole cell lysate at 20 µg

Lanes 5 and 10:

SARS-CoV 3xFlag Spike Protein Transfected Expi whole cell lysate at 20 µg

Secondary

Lanes 1 - 5:

Western blot - Donkey anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/donkey-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216778'>ab216778</a>) at 1/20000 dilution

Lanes 6 - 8:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/20000 dilution

Lanes 9 - 10:

Western blot - Donkey anti-Goat IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/donkey-goat-igg-h-l-irdye-800cw-preadsorbed-ab216775'>ab216775</a>) at 1/20000 dilution

Observed band size: 120-180 kDa,120-160 kDa,180 kDa

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