Anti-STAT3 Rabbit mAb - ChIP, CUT&RUN and CUT&Tag Grade

Overview

Description

Rabbit monoclonal antibody to STAT3

Reactivity

Mouse, Rat, Human

Tested applications

ICC/IF	: Use at an assay dependent dilution.
IHC-Fr	: Use at an assay dependent dilution.
IP	: Use at an assay dependent dilution.
WB	: 1:1000. Predicted molecular weight: 88 kDa.

 ChIP, CUT&RUN and CUT&Tag

Properties

Immunogen

Synthetic peptide (Mouse) (C terminal).

Clonality

Monoclonal, clone number: 5DF7

Isotype

IgG

Form

Liquid
PBS with 0.1% sodium azide

Storage instruction

Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.

Applications

WB Image

Anti-STAT3 antibody (AT0358) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size : 88 kDa
Observed band size : 90 kDa
Additional bands at : 38 kDa,40 kDa. We are unsure as to the identity of these extra bands.

Why is the actual band size different from the predicted?

 

Western blotting is a technique that separates proteins based on size - in general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors and so the actual band size observed may differ from that predicted. Common factors include...

• post-translational modification - e.g. phosphorylation, glycosylation etc which increases the size of the protein
• post-translation cleavage - e.g. many proteins are synthesized as pro-proteins, and then cleaved to give the active form, e.g. pro-caspases
• splice variants - alternative splicing may create different sized proteins from the same gene
• relative charge - the composition of amino acids (charged vs non-charged)
• multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands

ICC/IF Image

Cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/20 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted.