Far-western analysis was performed as described previously [32 (link)]. Briefly, proteins (50 pmol of all NEIL1 constructs, bovine serum albumin, and glutathione S-transferase; 10 pmol mtSSB) were separated by 12% SDS-PAGE, transferred to a PVDF membrane (BioRad), treated with 6M guanidine HCl in PBS containing 1 mM DTT, and then gradually refolded with successive dilutions of guanidine HCl in PBS containing 1 mM DTT. The membranes were then blocked for 3 hr with 3% BSA in PBS, 0.05% Tween 20 at 4 °C and incubated with pre-prepared HCT-116/HEK-293T whole cell extracts (1 mg/ml) or purified protein (10 pmol/ml), overnight. Immunoblot analysis was performed using an anti-SSB antibody (Abcam- ab74710).
Ab74710
Manufactured by Abcam
Anti-Actin antibody (ab74710) is a rabbit polyclonal antibody that recognizes actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. The antibody is suitable for use in various applications, such as Western blotting and immunocytochemistry, to detect and visualize actin expression.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (1 mentions)
Ab14715, by Abcam (1 mentions)
Ab14745, by Abcam (1 mentions)
Ab14705, by Abcam (1 mentions)
Ab14744, by Abcam (1 mentions)
Ab110242, by Abcam (1 mentions)
Ab14714, by Abcam (1 mentions)
Ab97505, by Abcam (1 mentions)
Ab110246, by Abcam (1 mentions)
Ab110273, by Abcam (1 mentions)
Ab56560, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
2 protocols using ab74710
1
Far-western analysis of NEIL1 protein interactions
2
Protein Expression Analysis in Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein gel electrophoresis and blotting analyses were performed on whole cell protein extracts obtained from patient (P) and control (C1 and C2) fibroblasts. Samples containing 30 μg protein were separated by denaturing NuPAGE 4%–12% Bis-Tris gels and transferred to nitrocellulose membrane. Immunodetection was carried out using primary antibodies against target proteins: RNase H1 (ab56560, Abcam), POLG (sc-5931, Santa Cruz), POLG2 (LS-C334882, LSBio), TWNK (gift from M Falkenberg), SSBP1 (ab74710, Abcam), TFAM (gift from RJ Wiesner), POLRMT (ab32954, Abcam), LRPPRC (ab97505, Abcam), SLIRP (ab51523, Abcam), ATAD3 (gift from JE Walker), bL12 (14795-1-AP, Proteintech), uL11 (SAB2701374, Sigma), MDDX28 (ab70821, Abcam), mS35 (16457-1-AP, Proteintech), mS18b (16139-1-AP, Proteintech), NDUFS3 (ab110246, Abcam), NDUFB8 (ab110242, Abcam), SDHA (ab14715, Abcam), SDHB (ab14714, Abcam), UQCRC1 (ab96333, Abcam), UQCRC2 (ab14745, Abcam), MT-CO1 (ab14705, Abcam), MT-CO2 (ab91317, Abcam), COX4l1 (ab14744, Abcam), ATPF1 (ab84625, Abcam), and ATPA1 (ab110273, Abcam), along with GAPDH (ab8245, Abcam), used as loading control. For quantifications, images were digitalized and analyzed with ImageJ software, and data analyses were performed in Microsoft Excel.
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