Ab99632

Manufactured by Abcam

Sourced in United Kingdom

Ab99632 is a rabbit monoclonal antibody that recognizes the human protein GAPDH. It is suitable for use in Western blotting, immunohistochemistry, and ELISA applications.

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Lab products found in correlation

4 protocols using ab99632

Antibody Characterization for Influenza A

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Primary antibodies used in this study include rabbit anti-KAP1 antibody (ab10484; Abcam), mouse anti-KAP1 antibody (ab22553; Abcam), rabbit anti-Phospho KAP1 (S824) antibody (A300-767A, Bethyl); mouse anti-β-actin antibody (A2228; Sigma-Aldrich, Darmstadt, Germany); mouse anti-FLAG antibody (F1084, Sigma-Aldrich), rabbit anti-influenza A NS1 polyclonal antibody purchased from GeneTex, Inc. (GTX125990 Q-1; Irvine, CA, USA). Mouse anti-M1antibody and mouse anti-HA antibody, mouse anti-NP, rabbit anti-NS2, rabbit anti-PB2, rabbit anti-PB1, rabbit anti-PA, mouse anti-M2, and rabbit anti-WSN virus antibodies were available in our laboratory. Secondary antibodies were used as follows: HRP-conjugated sheep anti-mouse IgG (GE Healthcare, NA931), HRP-conjugated donkey anti-rabbit IgG (GE Healthcare, NA934), HRP-conjugated Clean-blot IP detection reagent (Thermo Fisher Scientific, Waltham, MA, USA, 21230). HRP-conjugated rat monoclonal (H139-52.1] anti-mouse kappa light chain (Abcam, ab99632) and HRP conjugated anti-mouse IgG (Abcam, ab131368) secondary antibodies for IP blot.

Feng H., Yi R., Wu S., Wang G., Sun R., Lin L., Zhu S., Nie Z., He Y., Wang S., Wang P., Shu J, & Wu L. (2022). KAP1 Positively Modulates Influenza A Virus Replication by Interacting with PB2 and NS1 Proteins in Human Lung Epithelial Cells. Viruses, 14(4), 689.

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Investigating YAP1 Promoter Regulation

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For chromatin immunoprecipitation (ChIP), the promoter region of YAP1 was defined within 2000 base pairs upstream of the transcription start site and the sequence was obtained from the UCSC Table Browser [46 (link)]. Based on TRANSFAC (version 8.3), two putative binding sites of c-Jun were predicted at the region [47 (link), 48 (link)]. Crosslinking, lysis, and chromatin shearing were performed as previously described [25 (link)]. Targeted fragments (300–500 base pairs) were pulled down by Magnetic Dynabeads Protein G (1004, Life Tech), with c-Jun antibody or Normal IgG antibody. Primers for binding site sequences are listed in Supplementary Table S5. For co-immunoprecipitation, 500 µg cell lysate was used for immunoprecipitation with 20 µl magnetic beads, bonding with YAP1 antibody or normal IgG antibodyA light chain-specific secondary antibody (ab99632, Abcam) were used to avoid the signal of the heavy chain (~50 kDa).

Zhang J., Wong C.C., Leung K.T., Wu F., Zhou Y., Tong J.H., Chan R.C., Li H., Wang Y., Yan H., Liu L., Wu W.K., Chan M.W., Cheng A.S., Yu J., Wong N., Lo K.W., To K.F, & Kang W. (2020). FGF18–FGFR2 signaling triggers the activation of c-Jun–YAP1 axis to promote carcinogenesis in a subgroup of gastric cancer patients and indicates translational potential. Oncogene, 39(43), 6647-6663.

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Western Blot Analysis of Exosomal Proteins

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Isolated cells and exosomes were subjected to RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with the Protease Inhibitor Cocktail (Roche). Purified proteins were separated in 6%, 10%, or 12% SDS-PAGE (120 V for stacking gel and 160 V for separation gel) and then transferred to a nitrocellulose membrane in an ice bath. The nitrocellulose membrane was blocked with 5% bovine serum albumin for 1 h and then incubated overnight with primary antibodies at 4 °C. Antibodies used were mouse anti-CD63 (Abcam, ab59479), rabbit anti-CD9 (Abcam, ab92726), mouse anti-TSG101 (Santa, sc-7964), rabbit anti-GM130 (Abcam, ab30637), rabbit anti-Cltc (Cell Signaling Technology, #4796), rabbit anti-GAPDH (Abcam, ab181602). The membrane was then incubated with secondary antibodies (rat anti-mouse (Abcam, ab99632), mouse anti-rabbit (Abcam, ab99702)) for 1 h at room temperature and visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire UK).

Wan Z., Zhao L., Lu F., Gao X., Dong Y., Zhao Y., Wei M., Yang G., Xing C, & Liu L. (2020). Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium. Theranostics, 10(1), 218-230.

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Western Blot Analysis of RNA-Binding Proteins

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All samples were incubated at 95 °C for 10 minutes, separated on a 4–12% polyacrylamide Bis-Tris protein gel (NP0336BOX, Thermo), and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The membranes were blocked in 5% w/v milk (170–6404, Bio-Rad), 20 mM Tris-Cl (pH 7.6), 150 mM NaCl, and 0.1% v/v Triton X-100. The blots were probed with 0.7 μg/mL anti-RNAPII S5P antibody (ab5131, Abcam), 1 μg/mL anti-RNAPII S2P H5 antibody (920204, BioLegend), 0.365 μg/mL anti-hnRNPG antibody (ab190352, Abcam), 1 μg/mL anti-DDDDK (anti-FLAG) antibody (ab1162, Abcam), 0.23 μg/mL anti-METTL3 antibody (15073–1-AP, Proteintech), 1/1000 v/v anti-GST antibody (2624, Cell Signaling Technology), 1/3000 v/v anti-SNRP70 antibody (ab83306, Abcam), 1/5000 v/v anti-H3K4me3 (ab8580, Abcam), or 0.05 μg/mL anti-GAPDH antibody (A00192–40, Genscript), followed by 0.5 μg/mL mouse anti-rabbit light chain (ab99697, Abcam), 0.03 μg/mL goat anti-rabbit IgG (ab97051, Abcam), 1/2000 v/v rat anti-mouse light chain (ab99632, Abcam), or 0.05 μg/mL goat anti-mouse IgG antibody conjugated to horseradish peroxidase (ab97023, Abcam).

Zhou K.I., Shi H., Lyu R., Wylder A.C., Matuszek Ż., Pan J.N., He C., Parisien M, & Pan T. (2019). Regulation of co-transcriptional pre-mRNA splicing by m6A through the low-complexity protein hnRNPG. Molecular cell, 76(1), 70-81.e9.

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