HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Twist-1 Polyclonal Antibody (STJ31126)

What was used?Primary antibody: Twist-1 Polyclonal antibody (STJ31126)

Provider: St John's Laboratory

Dilution ratio: 1:500

Application: Western Blot

Materials for validation: HCT116 cells

Click for product data sheet PDF

What was the protocol?Treatment of Materials Cell pellet was lysed in 1.3 mls of 1 X 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 7.5% and then transferred and blotted

Gel Electrophoresis 10% polyacrylamide gel, constant voltage 90V, 120 min. 50 ug per lane.

Transfer Nitrocellulose membrane

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

Primary Antibody Application 1:500 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 2 hr.

Visualisation ECL kit

What was the protocol?Gel electrophoresis information: 10 % polyacrylamide gel, constant voltage 90 V for 60 min.

Transfer information: PVDF membrane, 1 piece of gel (semi-dry transfer) at 260 mA for 3 hours

Gel No. Antigen Loading amount

Primary antibody

Primary antibody

dilution ratio

Secondaryantibody

dilution ratioTarget band kDa Visualization

time

1 Mouse colon lysates, Wild type 50 ug

Twist-1 Antibody (STJ31126)

As indicated

1:7500 ~ 20 kDa See above

2Mouse colon

lysates, Rassf1a-/-

50 ug

3 HCT116 cells 10 ug

What were the results?(St John’s Laboratory)

“Appears to recognize both mouse and human forms on twist-1 in colon lysates and total cell lysates from HCT116 cells.”

What did the customer think?

Antibody Specificity:

Antibody Rating:

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

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