HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Phospho-AKT (S473) Polyclonal Antibody (STJ90166)

What was used?Primary antibody: Phospho-Akt (S473) Polyclonal antibody

(STJ90166)

Provider: St John's Laboratory

Dilution ratio: 1:1000

Application: Western Blot

Materials for validation: Mouse liver/colon lysates

Click for product data sheet PDF

What was the protocol?Treatment of Materials Cells were lysed in 1.5 mls of 1X standard RIPA lysis buffer with protease

inhibitors (PMSF and aprotinin) added. After lysis, samples were spun down and 4X sample buffer added. About 35 ul of the lysed cells was separated by SDS-PAGE at 10% and then transferred and blotted

Gel Electrophoresis 10% polyacrylamide gel, constant voltage 120V, 80 min. 50 ug per lane.

Transfer Nitrocellulose membrane

Blocking 1X TBST with 10% skimmed milk powder for 45 minutes.

Primary Antibody Application 1:1000 primary antibody was added to the solution of 1X TBST with 2.5% skimmed milk powder, 4˚C for overnight.

Secondary Antibody Application Secondary antibody (anti-rabbit HRP) was added to the solution of 1X TBST with 2.5% skimmed milk powder with 1:7500 dilution ratio, for 1.5 hours.

Visualisation ECL kit

What was the protocol?Gel electrophoresis information: 10 % polyacrylamide gel, constant voltage 90 V for 90 min.Transfer information: nitrocellulose membrane, 1 piece of gel (semi-dry transfer) at 260 mA for 3 hours

Gel No. Antigen Loading amount Primary antibody Primary antibody

dilution ratioSecondary

antibody dilution ratio

Target band kDa Visualization time

1 Mouse ColonWild type, no Tx 50 ug Phospho-Akt

(S473) antibody (STJ90166)

As indicated

1:7500 50 kDa 20min – 1 hour

2 Mouse ColonWild type, 3% DSS 50 ug 1:7500 50 kDa

20min – 1 hour

3,4 Mouse ColonRassf1a-/-, no Tx 50 ug

Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa

20min – 1 hour

5, 6 Mouse ColonRassf1a-/-, 3% DSS 50 ug

Phospho-Akt (S473) antibody

(STJ90166)

As indicated 1:7500 50 kDa

20min – 1 hour

7.8Mouse liver

Rassf1a-/-, 3% DSS + Metformin

50 ug Phospho-Akt (S473) antibody (STJ90166) As indicated 1:7500 50 kDa

20min – 1 hour

What were the results?

“The results demonstrate that the pS473 Akt can specifically recognize activated Akt in mouse samples. Most likely it will recognize human Akt. Antibody appears quite good.”

Testing results were provided independently by Professor Shairaz Baksh at the University of Alberta. They stated that:

What did the customer think?

Antibody Specificity:

Antibody Rating:

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