Anti-phospho-GSK3α/β (Ser21/9) (#9331) and anti-GAPDH (#2118) (Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-GSK3α/β (Y279/Y216) (#ab68476, Abcam, Cambridge, UK) were used in a 1:1000 dilution in TBS containing 3% BSA (PAA Laboratories, Pasching, Austria). Anti-active-β-Catenin (#05-665) (Merck Millipore, Billerica, MA, USA) was used in a 1:2000 dilution in TBS and 3% skim milk (Carl Roth, Karlsruhe, Germany). The species specific secondary antibodies anti-mouse IgG (whole molecule)–peroxidase (# A9044) and anti-rabbit IgG (whole molecule)–peroxidase (#A0545) (Sigma-Aldrich, St. Louis, MO, USA) were used in a 1:50.000 dilution in TBS containing either 3% BSA or 3% skim milk.
Skim milk
Manufactured by Carl Roth
Sourced in Germany
Skim milk is a dairy product that is obtained by removing the cream from whole milk. It contains less fat and calories compared to whole milk while retaining essential nutrients such as protein, calcium, and vitamins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg whole molecule peroxidase, by Merck Group (1 mentions)
Anti phospho gsk3α β ser21 9, by Cell Signaling Technology (1 mentions)
Anti mouse igg whole molecule peroxidase, by Merck Group (1 mentions)
Anti phospho gsk 3α β y279 y216, by Abcam (1 mentions)
Anti active β catenin, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Pierce precise protein gels, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti vsv m, by Kerafast (1 mentions)
Pbs t, by Carl Roth (1 mentions)
Bovine serum albumin (bsa), by AppliChem (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Mab911, by R&D Systems (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Complete mini, by Roche (1 mentions)
6 protocols using skim milk
1
Western Blot Analysis of Phospho-GSK3α/β
2
VACV Infection Impact on HEp-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot analysis, HEp-2 cells were infected with VACVNYCBOH at an MOI of 0.2 and cultivated for 3 to 4 days. Cells were lysed using RIPA lysis buffer supplemented with the HALT Protease Inhibitor cocktail (Thermo Fisher Scientific) and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). A protein lysate from mock-infected HEp-2 cells was used as the negative control. Proteins were separated on 8 to 16% precast gradient PAA gels (Pierce Precise™ Protein Gels, Thermo Fisher Scientific) and blotted onto a 0.2 μm PVDF membrane (VWR, Darmstadt, Germany). A PageRuler™ pre-stained standard (Fermentas, St. Leon-Rot, Germany) was used as the molecular weight marker. Membranes were blocked in TBS-T supplemented with 5% skim milk (Carl Roth) at 4°C overnight. Primary antibodies were incubated for 1 h at RT diluted in TBS-T supplemented with 1% skimmed milk, as were HRP-labelled species-specific secondary antibodies. Detection was done via chemiluminescence after 5 min incubation with ECL western blotting substrate (Thermo Fisher Scientific).
3
Western Blot Antibody Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used as primary antibodies: anti-V5 (mouse, 1:1,000; Thermo Fisher Scientific), anti-β-actin (ACTB, mouse, 1:1,000; Sigma-Aldrich), and anti-VSV-M (mouse, 1:1,000; Kerafast). As a secondary antibody, an HRP-coupled anti-mouse antibody was used (goat, 1:5,000; Dianova). All antibodies were diluted in PBS-T (Carl Roth) and 5% skim milk (Carl Roth).
4
H. pylori Infection and CagA Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 7.5 × 104 in a 24-well plate, two days before infection. On the day of the infection, one well was trypsinized and harvested cells were counted.
H. pylori grown on WC Dent plates for two days were collected, resuspended in BHI medium, and counted based on their optical density (OD600), with OD600 = 1 = 2 × 108 bacteria. The number of bacteria required for a multiplicity of infection (MOI) of 50 was determined, added to the eukaryotic cells and incubated for 6 h at 37 °C /5% CO2. After infection, the cells were washed 1x with PBS, lysed with Laemmli buffer + 50mM DTT, and run in an SDS-PAGE on 6% polyacrylamide gels followed by a Western blot onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The Western blot membrane was blocked with 5% skim milk (Carl Roth, Karlsruhe, Germany)/PBST probed with mouse anti-pTyr (1:300), rabbit anti-CagA serum (1:3000), and rabbit anti-GAPDH (1:1000) or anti-alpha tubulin (1:1000, loading control) in 5% Bovine serum albumin (Applichem, Darmstadt, Germany)/PBST. The CagA blot was reprobed with the anti-pTyr antibody to detect phosphorylated CagA.
H. pylori grown on WC Dent plates for two days were collected, resuspended in BHI medium, and counted based on their optical density (OD600), with OD600 = 1 = 2 × 108 bacteria. The number of bacteria required for a multiplicity of infection (MOI) of 50 was determined, added to the eukaryotic cells and incubated for 6 h at 37 °C /5% CO2. After infection, the cells were washed 1x with PBS, lysed with Laemmli buffer + 50mM DTT, and run in an SDS-PAGE on 6% polyacrylamide gels followed by a Western blot onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The Western blot membrane was blocked with 5% skim milk (Carl Roth, Karlsruhe, Germany)/PBST probed with mouse anti-pTyr (1:300), rabbit anti-CagA serum (1:3000), and rabbit anti-GAPDH (1:1000) or anti-alpha tubulin (1:1000, loading control) in 5% Bovine serum albumin (Applichem, Darmstadt, Germany)/PBST. The CagA blot was reprobed with the anti-pTyr antibody to detect phosphorylated CagA.
5
Quantifying Myocardial MMP-9 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cardiac infarct area was excised and lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) supplemented with a protease inhibitor cocktail (Complete Mini, Roche) using a tissue homogenizer (TissueLyser LT, Qiagen). Aliquots (30 μg) of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking for 1 h in Trisbuffered saline containing 0.1% Tween 20 and 5% skim milk (Carl Roth), membranes were probed overnight at 4°C with primary antibody against MMP-9 (R&D, MAB911; 1/2000 dilution) or 60 min for GAPDH (Sigma-Aldrich, G954; 1/10000). Densitometric quantification of protein bands was performed using ImageJ software (NIH, Bethesda, MD, USA) and MMP9 expression was normalized to GAPDH.
6
Protein Analysis in Aortic Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from aortic lysates was extracted using a Norgen Biotek kit according to the manufacturer's protocol (Catalog number: 48200, Norgen Biotek Corp). Aliquots of total protein were then size-fractioned by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking for 1 hour in Trisbuffered saline containing 0.1% Tween 20 and 5% skim milk (Carl Roth), membranes were incubated with primary antibodies against VCAM-1 (vascular cell adhesion molecule 1), MerTK (proto-oncogene tyrosine-protein kinase), or SR-B1 (scavenger receptor class B member 1) followed by detection with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Target protein expression was normalized to glyceraldehyde-3-phosphate dehydrogenase to correct for loading.
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!