Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody

Introduction

TAP-L (TAP-Like), also known as ABCB9, is an ATP-dependent membrane half-transporter. It belongs, like TAP, the transporter associated with antigen processing, to the ABC transporter family, the members of which transport various molecules across membranes. TAP-L can form homodimers and is located primarily in lysosomes, presumably importing peptides from the cytosol. TAP-L has broad specificity for peptides ranging from a length of 6 to 59 amino acids, with an optimal activity for peptides of 23 residues (Wolters et al., 2005). TAP-L can transport two peptides at a time (Herget et al., 2009). Considering its similarity to the heterodimeric TAP transporters (ABCB2/3) importing MHC class I peptide ligands into the endoplasmic reticulum, TAP-L is a potential candidate involved in antigen presentation by MHC molecules (Bangert et al., 2011). Indeed, the length of the peptides transported by TAP-L (6-59 residues) is compatible with loading of both MHC class I and class II molecules. Moreover, TAP-L is highly expressed in lysosomes of professional antigen presenting cell (APC) lysosomes, and upregulated during differentiation of dendritic cells. However, such a function remains hypothetical, and the biological role of TAP-L is presently unknown.

In this article, we describe experimentation designed to specifically detect the ABCB9 protein in bone marrow-derived dendritic cells (BMDCs) by immunoblot. We purchased a rabbit polyclonal antibody generated by Abcam Company using a synthetic peptide as the immunogen, corresponding to a region between residues 475 and 525 of human ABCB9. This antibody is expected to recognize mouse and human ABCB9 and recommended for immunohistochemistry (IHC), immunoprecipitation (IP) and western blot (WB).

Materials and methods

Results

Seeking to detect the ABCB9 protein, we performed a series of WBs on whole-cell lysates obtained from BMDCs, thought to correspond to an inflammatory subtype of DCs. It has previously been shown that ABCB9 expression by monocyte-derived human DCs is increased under inflammatory conditions (Demirel et al., 2007). To validate specificity of antibody staining, we included TAP-L deficient BMDCs as a negative control. TAP-L KO/WT heterozygous mice (ABCB9^{tm1 (KOMP) Vlcg}), in which the region located between nucleotides 5625 and 33216 of the TAP-L gene has been removed for the insertion of a cassette of 6085bp containing the cDNA conferring resistance to neomycin (Neo), were purchased from The Komp Repository at the University of California at Davis, CA 95616 (see construction of the KO gene; Figure 2A). Heterozygous mice were bred in our laboratory and inter-crossed to obtain homozygous KO mice (TAP-L KO/KO). To our surprise, the ABCB9 antibody recognized a band, with an apparent molecular weight (84kDa) corresponding to that of ABCB9 protein, both in WT and TAP-L deficient BMDCs (Figure 1). Three different immunoblots were performed in three independent experiments.

Figure 1. WB anti-ABCB9 on total cell lysates from WT and TAP-L KO BMDCs

20–200μg of total BMDC cell lysate from WT and TAP-L KO BMDCs was loaded on 10% acrylamide gels. The proteins were transferred onto a PVDF membrane. The rabbit ABCB9 antibody was used to detect the TAP-L protein (84kDa), followed by incubation with an HRP-conjugated goat anti-rabbit secondary antibody. An ECL detection system was used for developing the membranes by chemoluminescence. Three immunoblots from three independent experiments are shown.

Figure 2.

Strategy for genomic invalidation of the TAP-L/ABCB9 gene (A) and genotyping of WT and TAP-L deficient mice (B). In B, multiple KO mice were tested in the PCRs amplifying the Neo cassette and introns 5, 6 and 9. L, DNA ladder (See the results section for details).

Given these surprising results, we verified that the TAP-L KO mice were truly deficient for the target gene. We performed a series of polymerase chain reactions (PCRs). Different fragments of the WT allele (located in exons 2, 4, 8, 11 and introns 5, 6, 9) and the expected genomic region in KO mice (located between the upstream or downstream arm and within the Neo cassette) were amplified by PCR.

The following forward (F) and reverse (R) primers were used:

- Ex1-F: 5'-GTAGTAGTGACGCTGGCCTT-3' and Ex1-R: 5'CTTCTGTAGTGTGGCTCCCG-3', located in exon 1 of the WT allele and amplifying a product of 498bp in the WT allele

- Ex2-F: 5'-AGACCTTCCTGCCCTACTACA-3' and Ex2-R: 5'-CAGCAGGCAAACGACGACAA-3', located in exon 2 of the WT allele and amplifying a product of 101bp in the WT allele

- Ex4-F: 5'-CGCCTCACCTCTGATACCAC-3' and Ex4-R: 5'-TGCCGTAGATGTTGGACACC-3', located in exon 4 of the WT allele and amplifying a product of 181bp in the WT allele

- Ex8-F: 5'-CAAGGTGACAGCTCTGGTGG-3' and Ex8-R: 5'-GCCATCCAACAATACACGGC-3', located in exon 8 of the WT allele and amplifying a product of 106bp in the WT allele

- Ex11-F: 5'-GAGACACACGGTGCTCATCA-3' and Ex11-R: 5'-TGTGTTCAGTGTTGCTGGGT-3', located in exon 11 of the WT allele and amplifying a product of 214bp in the WT allele

- INT5-F: 5'-TACTCGGGTGCCACTACCTG-3' and INT5-R: 5'-GGCACATGCCACCTTCAAGT-3', located in intron 5 of the WT allele and amplifying a product of 379bp in the WT allele

- INT6-F: 5'-TGCTTAAAGGCACTCGGTGA-3' and INT6-R: 5'-CTTCGGGGATACCACAGAGC-3', located in intron 6 of the WT allele and can amplifying a product of 371bp in the WT allele

- INT9-F: 5'-TGCCAAGTTTAGTGCCAGGATG-3' and INT9-R: 5'-GCCCAGGACAAAAAAAGCAATC-3', located in the intron 9 of the WT allele and amplifying a product of 371bp in the WT allele

- KOFwd1: 5'-TTGCATGGAGAAGACCCTCC-3', located in the arm upstream of the Neo cassette (Neo upstream arm), and KORvs1: 5'-GAGGGGACGACGACAGTATC-3', located in the Neo cassette and amplifying a product of 465bp in the KO allele

- KOFwd2: 5'-GCAGCCTCTGTTCCACATACACTTCA-3', located in the Neo cassette and KORvs2: 5'-GCTTAGTTCTCTCCCAGACATCCTCC-3', located in the arm downstream of the Neo cassette (Neo downstream arm) and amplifying 425bp in the KO allele.

PCRs were performed in a total volume of 25μl containing: 17.3μl H2O (DEPC treated water, pathogen free, DNase/RNase Free-Invitrogen), 5 μl 5x GoTaq Green Reaction Buffer (Promega), 0.5μl dNTP (10mM), 20 μM primers, 0.2μl polymerase (5 U/μl) (GoTaq-Promega polymerase) and 1μl DNA or water (negative control). The amplification reaction was performed as follows: for the WT allele, an initial denaturation at 94°C for 5 min, 10 cycles: denaturation 94°C for 15 sec, annealing 65°C for 30 sec, elongation 72°C for 40 sec; 30 cycles denaturation 94°C for 15 sec, annealing 55°C for 30 sec, elongation 72°C for 40 sec; final elongation 72°C for 5 min. For the KO allele: initial denaturation at 94°C for 5 min; 10 cycles: denaturation 94°C for 15 sec, annealing 62°C for 30 sec, elongation 72°C for 40 sec; 25 cycles: denaturation 94°C for 15 sec, annealing 57°C for 30 sec, elongation 72°C for 40 sec; final elongation 72°C for 5 min. The PCR products obtained were migrated on a 1.5% agarose gel containing 10 μg/ml of Ethidium Bromide. Migration was performed in a buffer tank filled with TAE buffer containing 40mM Tris, 20mM acetic acid, 1mM EDTA, pH=8 for 20 min at 120 V and visualization of the PCR products under a UV lamp connected to a photographic device.

The resulting PCR products from multiple KO mice confirmed the absence of the TAP-L gene and the presence of the Neo cassette (Figure 2B), indicating that the TAP-L gene was deleted as expected and that the mice obtained were TAP-L KO/KO. Consequently, the band recognized by the ABCB9 antibody, even though running at the expected molecular weight, could not correspond to the TAP-L protein.

Conclusion

Collectively, these results show that the commercial ABCB9 antibody recognizes a protein with a molecular weight similar to that of TAP-L. It is impossible to know whether it also recognizes TAP-L. Our findings highlight the importance of verifying commercial antibody specificity using knockout cells. If such cells are not available, lentiviruses encoding target-specific shRNA, which are now readily available for an essentially complete range of proteins, can be used to produce cells that provide informative negative controls.