Anti-Stathmin 1 (phospho S63) antibody [EPR1574]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

ab76583, at a 1/250 dilution, staining Stathmin 1 in paraffin embedded human brain tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

Stathmin 1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) treated with Calyculin A whole cell lysate 10 μg with ab76583 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with Calyculin A whole cell lysate 10 μg

Lane 2 : ab76583 IP in HeLa treated with Calyculin A whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab76583 in HeLa treated with Calyculin A whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

75kDa band could be stathmin/alpha tubulin complex. (PMID : 9369201)

All lanes:

Immunoprecipitation - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (ab76583)

Predicted band size: 17 kDa

Observed band size: 17 kDa

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• WB

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Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

All lanes:

Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (ab76583) at 1/10000 dilution

Lane 1:

HeLa cell lysate, untreated at 10 µg

Lane 2:

HeLa cell lysate treated with Calyculin A at 10 µg

Secondary

All lanes:

HRP labelled goat anti rabbit at 1/2000 dilution

Predicted band size: 17 kDa

Observed band size: 17 kDa

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• WB

Lab

Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

Blocking/Diluting buffer and concentration 2% BSA/TBST

All lanes:

Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (ab76583) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates at 15 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with nocodazole at 100 ng/mL for 18 hours. Whole cell lysates at 15 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with nocodazole at 100 ng/mL for 18 hours. Whole cell lysates. Then the membrane was incubated with phosphatase. at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 17 kDa

Observed band size: 17 kDa

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Exposure time: 30s

• WB

AbReview27604****

Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

Alkaline phosphatase treatment removes S63 phosphorylation. Samples treated with phosphatase were run alongside the normal lysate, and the phosph-S63 signal is not detected after phosphatase treatment, thus suggesting the signal is very specific to the phosphorylated S63.

Whole cell lysate prepared from a human colon cancer cell line was loaded at 20μg.
ab76583 used at a 1/1000 dilution.
Secondary used was an HRP conjugated goat anti-rabbit polyclonal used at a 1/10000 dilution.

All lanes:

Western blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (ab76583)

Predicted band size: 17 kDa

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Image courtesy of Qifeng Lin by Abreview.

• Dot

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Dot Blot - Anti-Stathmin 1 (phospho S63) antibody [EPR1574] (AB76583)

Dot blot analysis of Stathmin 1 (phospho S63) phospho peptide (Lane 1) and Stathmin 1 non-phospho peptide (Lane 2) labeling Stathmin 1 (phospho S63) with ab76583 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/100 000 was used as the secondary antibody.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.