Purified rFIX samples were separated by SDS–PAGE using a 4–12% Bis-Tris Bolt acrylamide gel (Invitrogen) in 1× MES running buffer (Invitrogen), under nonreducing conditions, following manufacturer’s instructions. Protein bands were visualized either with colloidal Coomassie blue (Simply Blue; Invitrogen) or western blotting. For western blotting, proteins were transferred from gels onto a PVDF membrane (Bio-Rad) using a Trans-Blot® Turbo™ Transfer System Transfer Pack (Bio-Rad), following manufacturer’s instructions. The membrane was blocked in 2% skim milk (Devondale) dissolved in phosphate-buffered saline solution (Sigma) with 0.05% Tween 20 (Sigma) for 30 min at room temperature. The following antibodies were used: mouse IgG1 anti human FIX primary antibody 1C2 (AbCam, 1:1000 dilution) and HRP-conjugated anti mouse IgG (Bio-RAD, 1:1000 dilution). Membranes were developed with Novex ECL reagent (Invitrogen) and imaged using a Bio-Rad image workstation.
Trans blot turbo transfer system transfer pack
Manufactured by Bio-Rad
The Trans-Blot® Turbo™ Transfer System Transfer Pack is a laboratory equipment product designed for the rapid and efficient transfer of proteins from polyacrylamide gels to membranes. The transfer pack includes the necessary components for the transfer process, facilitating the transfer of proteins in a short duration of time.
Automatically generated - may contain errors
Lab products found in correlation
Image workstation, by Bio-Rad (1 mentions)
Phosphate buffered saline solution, by Merck Group (1 mentions)
Bis tris bolt acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Novex ecl reagent, by Thermo Fisher Scientific (1 mentions)
Simplyblue, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mini protein tgx gel, by Bio-Rad (1 mentions)
Multi gauge version 3, by Fujifilm (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Las 3000, by Fujifilm (1 mentions)
2 protocols using trans blot turbo transfer system transfer pack
1
SDS-PAGE and Western Blot Analysis of rFIX
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were extracted in RIPA buffer® according to the manufacturer's instructions (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Ten μg of cytosolic fractions were and loaded onto Mini-PROTEIN® TGX™ gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using Trans-Blot® Turbo™ Transfer System Transfer Pack (Bio-Rad Laboratories). Subsequently, the membranes were blocked for 1 hour in 4% bovine serum albumin (BSA) in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) and incubated overnight at 4°C in primary antibody in TBS-T with 4% BSA. The following antibodies and concentrations were used: 1/2,500 rabbit LC3A, 1/2,500 rabbit PARP, 1/2,500 rabbit cleaved-PARP, 1/5,000 rabbit β-actin. After 3 washes with TBS-T, membranes were incubated for 1 hour at room temperature using horseradish peroxidase-conjugated anti-rabbit secondary antibody as appropriate. After 3 washes with TBS-T, they were visualized using the ECL detection system (GE Healthcare UK Ltd., England, UK) by a LAS-3000 (Fujifilm, Tokyo, Japan). Protein expression was determined densitometrically and normalized against β-actin expression using Multi Gauge version 3.1 (Fujifilm).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!