Using WB, the primary antibody, rabbit polyclonal anti-MX1 antibody (ab95926, Abcam, UK), was validated. BC cell line lysate, MCF7, and human embryonic kidney (HEK) that was used as a control (from the American Type Culture Collection, Rockville, MD, USA) were employed for WB antibody specificity validation. MX1 antibody was used at a dilution of 1:1500 and IRDye 800CW Donkey anti-Rabbit fluorescent secondary antibody (LI-COR Biosciences) was used at a 1:15,000 dilution. For loading control, mouse monoclonal anti-β-actin primary antibody (1:5000, Sigma-Aldrich) was used and followed by incubation with anti-Mouse fluorescent secondary antibody (LI-COR Biosciences). To detect the protein molecular weight, 20 µg of the cell lysate was loaded alongside the protein ladder (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific). A specific band was detected at the predicted molecular weight of ~ 64 kDa using Odyssey Fc scanner and visualised by Image Studio 4.0 software (Supplementary Fig. 1).
Ab95926
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab95926 is a laboratory product offered by Abcam. It is a basic lab equipment item without any additional details about its core function or intended use.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti β actin primary antibody, by Merck Group (1 mentions)
Irdye 800cw donkey anti rabbit fluorescent secondary antibody, by LI COR (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Ab113112, by Abcam (1 mentions)
Anti stat1, by BD (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mab1501, by Merck Group (1 mentions)
A 21202 and a 21203, by Thermo Fisher Scientific (1 mentions)
Gapdh 14c10 number 2118, by Cell Signaling Technology (1 mentions)
Respective alexafluor 488 and alexafluor 594 tagged secondary donkey antibodies to rabbit, by Thermo Fisher Scientific (1 mentions)
A 11008 and a 11012, by Thermo Fisher Scientific (1 mentions)
Lumi light western blotting substrate, by Merck Group (1 mentions)
Alpha imager, by Bio-Techne (1 mentions)
A5316, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Sc 130568, by Santa Cruz Biotechnology (1 mentions)
Gtx125990, by GeneTex (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
5 protocols using ab95926
1
Validating MX1 Antibody Specificity by Western Blotting
2
Western Blot Analysis of Immune Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infected tissues were lysed with RIPA buffer at 2 dpi. 15μg of lysed proteins were loaded on a 10% SDS-PAGE gel and then transferred onto PVDF membrane (Bio-Rad). The membranes were first blocked with 5% skim milk (AppliChem) in TTBS (10 mM Tris HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20) at RT for 30min and then incubated with primary Ab overnight at 4°C. The membranes were then washed thrice with TTBS and incubated with secondary antibodies conjugated to HRP at RT for 1h. The membranes were washed and later developed with Western Bright Sirius ECL system (Advansta Inc.) for 2 min. Immunoblot images were acquired using Fujifilm LAS 4000 luminescence imager. Primary antibodies used in Western blot include anti-Stat1 (612233, BD), -MDA5 (ENZ-ABS299-0100, Enzo Life Sciences), -MX1 (ab95926, Abcam), -IFIT2 (ab113112, Abcam) and -actin (MAB1501, Merck Millipore). All primary antibodies were diluted 1:1000 except MX1 diluted in 1:2000. Goat anti-rabbit (7074, Cell Signaling Technology) and horse anti-mouse (7076, Cell Signaling Technology) secondary antibodies conjugated to HRP were diluted in 1:5000 and used for chemiluminescence development.
3
Immunofluorescence Antibody Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit pAb to human MxA (H-285) (ab-95926) was purchased from Abcam Inc. (Cambridge, MA, USA); Mouse mAb to the VSV nucleocapsid (N) designated 10G4 was a gift from Dr. Douglas S. Lyles (Wake Forest School of Medicine, Winston-Salem, NC, USA). Rabbit mAb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 14C10; number 2118) was obtained from Cell Signaling (Danvers, MA, USA), Anti-HA murine mAb (Cat. No. 100028-MM10) was purchased from Sino Biologicals US Inc. (Wayne, PA, USA). Respective AlexaFluor 488- and AlexaFluor 594-tagged secondary donkey antibodies to rabbit (A-11008 and A-11012) or mouse (A-21202 and A-21203) IgG were from Invitrogen Molecular Probes (Eugene, OR, USA). Alexafluor-594 tagged recombinant cholera toxin subunit B (CTB-red) was also purchased from Invitrogen Molecular Probes and used as per manufacturer’s recommendations (10 µM final concentration).
4
Immunoblotting Analysis of Influenza NS1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extracted from the cells were separated by SDS-PAGE and transferred onto PVDF membranes (Invitrogen). Anti-UAP56 antibody (ab1811061; abcam), anti-FLAG M2 antibody (F1804; Sigma), anti-β-actin antibody (A5316; Sigma), and anti-Mx1 antibody (ab95926; abcam) were used for immunoblotting. Wild-type (WT) and mutant WSN-NS1 proteins were analyzed with anti-influenza A NS1 antibody sc-130568 (Santa Cruz) and/or GTX125990 (GeneTex). Blots were developed using Lumi-Light Western blotting substrate (Sigma) or SuperSignal West Femto Maximum sensitivity substrate (Thermo), and exposed to X-ray film Super RX-N (FUJI film) or analyzed by AlphaImager (Alpha Innotech).
5
Immunoblot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblots were performed following standard procedures (25 (link)). Specific antibodies against LAMP2A (ab18528, Abcam, 1/500), SOX2 (AB5603, Millipore, 1/250), SOX9 (AB5535, Millipore, 1/1,000), TIM23 (11123–1-AP, Proteintech, 1/1,000), MRSP23 (SAB2701383, Sigma, 1/500), p-STAT1 (9167S, Cell Signaling, 1/1,000), STAT1 (14994S, Cell Signaling, 1/1,000), MX1 (ab95926, Abcam, 1/500), p-STAT3 (9145S, Cell Signaling, 1/1,000), STAT3 (9139, Cell Signaling, 1/1,000), ITGA6 (3750S, Cell Signaling, 1/250), ITGB4 (HPA036348, Sigma, 1/500), p-AKT (9271, Cell Signaling, 1/1,000), AKT (sc-8312, Santa Cruz Biotechnology, 1/200), and β-actin (A5441, Sigma-Aldrich, 1/100,000) were used in the study, performing overnight incubations at 4°C in orbital agitation. For secondary antibodies, horseradish peroxidase (HRP)-linked anti-rabbit (7074S, Cell Signaling, 1/2,000), or anti-mouse (7076S, Cell Signaling, 1/2,000) were used, performing incubations of 1 hour at room temperature in orbital agitation. Detection was performed by chemiluminiscence using NOVEX ECL Chemi Substrate and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Same β-actin were represented when analyzed samples were the same and have been treated in same conditions.
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!