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. 2024 Sep 5;37(9):1588–1597. doi: 10.1021/acs.chemrestox.4c00307

Sensitive Detection of Histones and γ-H2AX by Immunoblotting: Problems and Solutions

Casey Krawic †, Michal W Luczak ‡, Anatoly Zhitkovich †,*
PMCID: PMC11409373  PMID: 39237351

Abstract

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Histones and their posttranslational modifications (PTMs) are critical regulators of gene expression. Differentiation, environmental stressors, xenobiotics, and major human diseases cause significant changes in histone variants and PTMs. Western blotting is the mainstay methodology for detection of histones and their PTMs in the majority of studies. Surprisingly, despite their high abundance in cells, immunoblotting of histones typically involves loading of large protein amounts that are normally used for detection of sparse cellular proteins. We systematically examined technical factors in the Western-blotting-based detection of human histones with >30 antibodies. We found that under multiple protein transfer conditions, many histone epitopes on polyvinylidene fluoride (PVDF) membranes had a very low antibody accessibility, which was dramatically increased by the addition of a simple denaturation step. Denaturation of membrane-bound proteins also enhanced the specificity of some histone antibodies. In comparison to standard PVDF membranes, the sensitivity of histone detection on standard nitrocellulose membranes was typically much higher, which was further increased by the inclusion of the same denaturation step. Optimized protocols increased by >100-times detection sensitivity for the genotoxic marker γ-H2AX with two monoclonal antibodies. The impact of denaturation and nitrocellulose use varied for different histones, but for each histone, it was generally similar for antibodies targeting N-terminal and C-terminal regions. In summary, denaturation of membrane-bound histones strongly improves their detection by Westerns, resulting in more accurate measurements and permitting analyses with small biological samples.

Introduction

Histones are small nuclear proteins that are responsible for packaging genomic DNA into nucleosomes. Nucleosomes are formed by wrapping approximately 146 bp of DNA around a histone octamer composed of two copies of four core histones H2A, H2B, H3 and H4. Further compaction of nucleosome arrays is produced by histone H1 that binds linker DNA connecting individual nucleosomes.1,2 Histones are rich in positively charged amino acids Lys and Arg, which confer strong electrostatic binding to the negatively charged DNA phosphate backbone. Tight association of nucleosomal DNA with histones restricts its accessibility to transcription factors, which can be modulated by post-transcriptional modifications (PTMs) of core histones. Structures of all four core histones include central globular domains and long N-terminal tails with the latter containing the majority of amino acids undergoing PTMs.3,4 Histone PTMs include several different chemical modifications of the multiple amino acids. The importance of acetylation and methylation on the ε-amino group of specific lysines is the most well understood and these PTMs are the most frequently assessed on the global or locus-specific levels.5,6

Histone PTMs are linked to major physiological processes such as differentiation and development as well as pathogenesis of major human diseases, including cancer, cardiovascular disease and neurodegeneration.7−11 A widespread involvement of histone PTMs in cancer led to extensive efforts targeting them for drug development.12,13 Histone PTMs exhibit two-way interactions with cellular metabolism by regulating gene expression and being sensitive to the yield of specific products in central metabolic pathways.14,15 Environmental stressors such as hypoxia16,17 and various toxicants18−20 have also been shown to modulate the levels of histone PTMs. DNA damage, especially the formation of DNA double-stranded breaks, induces phosphorylation at Ser139 in histone H2AX which acts as a platform for the recruitment of DNA repair and chromatin remodeling factors.21,22 All of these roles of histone PTMs led to numerous publications which assessed their levels under normal, pathological, or stress-induced conditions. Mass-spectrometry methodologies are indispensable for analysis of a wide spectrum of small histone PTMs,23 however, these approaches require expensive equipment, trained personnel and involve complex workflows. The vast majority of chromatin studies are focused on changes in PTMs at a single or a small number of lysines or other amino acids, which are typically assessed by the workhorse methodology of Western blotting. Histones are the most abundant proteins in all nucleated cells, and it could be easily assumed that immunodetection of histones and their PTMs would require only small amounts of samples. However, immunoblots in many published studies were typically done with large amounts of total cellular proteins commonly used for detection of much less abundant regulatory proteins, and commercial vendors frequently demonstrate reactivity of their antihistone antibodies with Westerns using 10–50 μg of cellular histones. The need for large amounts of histones for immunoblotting is problematic when the availability of samples is limited such as in cases of rare cell populations obtained after cell sorting or from biopsies. Loading of large amounts of proteins is also associated with higher nonspecific signals and nonlinear responses.24,25 Mass use of antihistone antibodies led to systematic efforts to evaluate their specificity,26 whereas investigation of technical factors affecting detection of histones by immunoblotting was much more limited and tested only anti-H3 antibodies.27

In this work, we examined detection of all major histones and their multiple PTMs by immunoblotting using different conditions and testing >30 antihistone antibodies with whole cell lysates, chromatin extracts and recombinant histones. We found that a low detection sensitivity for several histones and their multiple PTMs was caused by a very poor epitope availability, especially on polyvinylidene fluoride (PVDF) membranes. Both sensitivity and for some antibodies, also specificity of histone detection can be dramatically increased by the addition of a simple protein-denaturation step after completion of protein transfer to PVDF or nitrocellulose membranes (NC). In general, nitrocellulose membranes gave a much higher sensitivity of detection for core and linker histones but not for typical nonhistone loading controls or other small proteins.

Experimental Procedures

Materials

Individual histones were purchased from New England Biolabs (H2A: M2502S, H2B: M2505S, H3.1: M2507S, H4: M2504S), Epicypher (H2AX: 15–0307) and Sigma (H2AZ: 14–1109). Bleomycin was obtained from LKT Laboratories (B4518), etoposide from Sigma-Aldrich (E1383), camptothecin from Sigma-Aldrich (C9911) and Trichostatin A from Cayman Chemical (89730).

Cell Culture

H460 and IMR90 human cells were obtained from the American Type Culture Collection. H460 cells were grown in RPMI-1640 medium (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum and penicillin/streptomycin. Primary IMR90 cells were propagated in DMEM medium (Gibco, 12430–062) containing 20% fetal bovine serum. Both cell lines were grown at 37 °C in the atmosphere of 95% air/5% CO2.

Preparation of Cell Lysates

Whole cell lysates were prepared by heating cells at 99 °C for 10 min in a 2% SDS solution (2% SDS, 50 mM Tris-HCl pH 6.8, 10% glycerol) containing Halt Protease and Phosphatase Inhibitors (ThermoFisher Scientific, PI78443). For isolation of insoluble nuclear material/chromatin, soluble proteins were first extracted using a cell lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5% NP40, 2.5% glycerol, and protease/phosphatase inhibitors) for 15 min on ice followed by centrifugation at 10 000g for 10 min at 4 °C. Pellets were solubilized using the 2% SDS lysis buffer and heating at 99 °C for 10 min. All protein samples were stored at −80 °C. Protein concentrations were measured using BioRad DC Protein Assay Reagents A (5000113), B (5000114) and S (5000115). For loading on gels, protein lysates were mixed with 5x loading buffer (10% SDS, 25% β-mercaptoethanol (added fresh), 50% glycerol, 250 mM Tris-HCl and 0.5% bromophenol blue, pH 6.8), heated for 10 min at 99 °C and then placed on ice.

Immunoblotting

Duplicate sets of samples were loaded onto 12% SDS-PAGE gels, and after the completion of electrophoresis, proteins were transferred onto 0.2 μm PVDF (Bio-Rad, 1620177) or 0.2 μm nitrocellulose (Bio-Rad, 1620112) membranes using semidry or overnight wet transfer procedures. PierceG2 Fast Blotter (13 min, 25 V, 2.5A) with 12% ethanol-supplemented transfer buffer (ThermoFisher, 84731) and Bio-Rad Trans-Blot Turbo Transfer System (10 min, 25 V, 2.5A) with 20% ethanol-containing transfer buffer were used as standard semidry procedures. In some experiments, semidry transfers with PierceG2 Fast Blotter used different buffers and time/electric settings as specified in Figure legends. Wet transfer onto PVDF membranes was done overnight at 18 V using cold 20% methanol-supplemented Towbin buffer (25 mM Tris, 192 mM glycine, pH 8.3) or Bjerrum Shaefer-Nielsen buffer (48 mM Tris, 39 mM glycine, pH 9.2). After transfer by any procedure, the membranes were cut in half (when two sets of samples were loaded, or more pieces if higher numbers of sample sets were loaded on gels): one-half was placed in 1x TBST buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20), and the other half was placed in boiling Milli-Q water for 30 min with light shaking. Boiled membranes were placed into room temperature water for about 30 s and then transferred to TBST. Both pieces of the membranes were washed 3x for 5 min in TBST at room temperature and then blocked in TBST with 5% milk for 1 h at room temperature. Membranes were washed 3x for 5 min at room temperature and then incubated with primary antibodies in TBST with 5% milk or 5% BSA at 4 °C overnight with shaking. Next day, membranes were washed 3x 5 min in TBST at room temperature and then incubated with secondary antibodies in TBST with 5% milk for 1 h with shaking at room temperature. After 3x 5 min washes in TBST at room temperature, both halves of the membranes were taped together and developed with the ECL Western Blotting Detection Reagent (RPN2232, GE Life Sciences). Primary and secondary antibodies were used at manufacturers’ recommended dilutions. Band intensities were quantified by using ImageJ.

Antibodies

Histone antibodies: H1 (Millipore, 05–457), H1.2 (GeneTex, GTX122561), total H2A (Cell Signaling, 12349 and 2578), H2A.1 (Millipore, ABE327), macroH2A1.2 (Cell Signaling, 4827), H2AX (Cell Signaling, 7631 and 2595 and Invitrogen, MA5–24663), γH2AX (Cell Signaling: 2577; Abcam: ab26350; Millipore: 05–636), H2AZ (Cell Signaling, 2718), H2Aub1 (Cell Signaling, 8240), H2AK5ac (Cell Signaling, 2576), H2B (Cell Signaling, 12364 and Millipore, 07–371), H2Bub1 (Cell Signaling, 5546), H2BK5ac (Cell Signaling, 12799), H3 (Cell Signaling, 9715), H3.3 (Novus, NBP2–24697), H3K9ac (Cell Signaling, 9649), H3K4me3 (Millipore, 07–473), H3K27me3 (Cell Signaling, 9733), H3K79me3 (EpigenTek, A-4045–050), H3K27ac (Cell Signaling, 9733), H4 (Cell Signaling, 13919 and Millipore, 07–108), H4K8ac (Cell Signaling, 2576), H4K12ac (Active Motif, 39165), H4K16ac (Abcam, ab109463) and H4K20me3 (Abcam, ab9053 and Cell Signaling, 5737). Other primary antibodies: RPA32 (Millipore, MABE285), TRX1 (Cell Signaling, 2429), ubiquitin (Cell Signaling, 58395), fibrillarin (Cell Signaling, 2639), GAPDH (Cell Signaling, 3683), L7A (Cell Signaling, 2415), β-actin (Cell Signaling, 4967), PCNA (Santa Cruz, sc56), and γ-tubulin (Sigma, T6557). Secondary antibodies were horseradish peroxidase-conjugated goat antimouse (Millipore, 12–349) and goat antirabbit (Cell Signaling, 7074).

Results

Detection of Cellular Histones with Standard and Boiled PVDF Membranes after Semidry Transfer

Apart from the quality of specific antibodies, the detection sensitivity of histones (and other proteins) by immunoblotting has largely been considered as a function of their efficient transfer from gels and the retention efficiency by the membranes. For small proteins such as histones, membranes with small pores (0.2 μm) in combination with semidry procedures using ethanol- or methanol-containing buffers offer fast transfers with sharp bands and a complete retention of proteins (as easily verified by using two membranes for transfers and then staining both of them and the gel for proteins).28−30 Two types of membranes, PVDF and nitrocellulose, are typically used for the immunoblotting of histones and other proteins. PVDF membranes offer some advantages such as easier handling, higher protein binding capacity, and better simultaneous detection of smaller and larger proteins. Based on a very limited comparison with NC membranes (probing for K56-acetylated H3), PVDF membranes were considered to be somewhat more sensitive for detection of histones.27 Therefore, we first examined histone detection using PVDF membranes. In consideration of the apparently low detection sensitivity of histones despite their high abundance, we focused on the possibility that antibody-binding epitopes on PVDF membrane-bound histones are poorly accessible, which could be exposed by denaturation. Our initial tests with chemical denaturation of PVDF-bound proteins (guanidine-HCl, NaOH or HCl) improved detection of some histones, but these treatments were too harsh for use on NC membranes. We next explored protein denaturation by boiling PVDF membranes after semidry transfer and testing the immunodetection of four core histones and four typical protein loading controls. We found that boiling of PVDF membranes for 10–30 min showed dramatically increased detection sensitivity for histones H4, H2B, H2A and K119-monoubiquitinated H2A (Figure 1A). H2A was probed with an antibody that was expected to detect three major variants of this histone: canonical H2A and much less abundant H2AX and H2AZ. In the nonboiled membranes, this antibody detected a band with a higher molecular weight than canonical H2A which in subsequent tests with purified histones was identified as H2AX. Boiled PVDF membranes revealed the presence of abundant canonical H2A while retaining the signal for the higher band, which is H2AX (Figure 1A). Detection of histone H3 and four nonhistone loading controls (γ-tubulin, L7A, GAPDH and fibrillarin) was not noticeably affected by boiling of PVDF membranes. Next, we examined how neutralization of positive charges on histone lysines through acetylation can affect detection of histones and their PTMs. H460 cells were treated with the histone deacetylase inhibitor Trichostatin A for 18 h, which caused massive increases in histone Lys acetylation at all of the analyzed sites (Figure 1A,B). Boiled PVDF membranes again showed highly increased sensitivity for detection of specific histones and their lysine acetylation: H4 (with a different antibody than in Figure 1A) and its acetylation at three sites; H2B and its K5 acetylation and K120 monoubiquitination; H2A (with all three tested antibodies) and its K5 acetylation and K119 monoubiquitination; and H2A variants H2AX and H2AZ (Figure 1B). In addition, boiling also eliminated a preferential recognition of H2AX with two anti-H2A antibodies that are expected to detect the most abundant canonical form of H2A with a slightly lower molecular weight. Hyperacetylation of H2AX and H2B also diminished the detection of their total levels, which remained equally high for control and TSA-treated samples on boiled membranes. In total, our tests with 16 antihistone antibodies showed a remarkedly higher detection sensitivity on the boiled PVDF membranes. For some antibodies, boiling also increased their specificity for the main targets (such as canonical H2A with pan-variant H2A antibodies). Control and hyperacetylated samples showed similar detection sensitivity for three loading controls (γ-tubulin, β-actin and L7A) and histones H3, H3.3 and H3 acetylated at K9 (Figure 1C). To exclude potential cell-type-specific effects, we next tested the impact of PVDF membrane boiling on detection of histones and their PTMs in whole cell lysates from IMR90 primary human cells using the same semidry transfer. Different amounts of proteins ranging from 0.3 to 10 μg/lane were loaded to compare the detection sensitivity of histones with standard and boiled PVDF membranes. Comparison of the band intensities in 1 and 3 μg lanes again showed a much greater sensitivity of boiled PVDF membranes for detection of all three H2A variants, monoubiquitinated H2A, and H2B probed with two antibodies (Figure 2A). In immunoblots for H4, all five tested antibodies also showed much higher sensitivity in boiled PVDF membranes (Figure 2B). Under exposure conditions when signals in 10 μg lanes on boiled membranes were already saturated, the majority of antibodies on standard PVDF membranes gave strong signals on 10 μg lanes but the detection sensitivity was abruptly lower in 3 ug lanes (Figure 2A,B). Thus, detection of H4, H2A and H2B histones and their PTMs on standard PVDF membranes can be enhanced in a supra-linear fashion by increasing protein loading but the nonlinear dependence of the signal on protein amounts adversely affects quantitation and specificity of Western blots.24,25 Similar to findings with H460 samples, standard and boiled PVDF membranes with IMR90 lysates showed a comparable detection sensitivity of loading controls (tubulin, actin, PCNA and L7A) and histone H3 and its three PTMs (Figure 2C).

Figure 1.

Figure 1

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Impact of PVDF membrane boiling on detection of cellular histones and their posttranslational modifications. Duplicate sets of samples were transferred on PVDF membranes using PierceG2 Fast Blotter and membranes were cut in four (A) or two (B,C) pieces. One piece underwent washes with TBST while others were boiled in water for the indicated periods of time before TBST washes. Whole cell lysates of H460 cells were analyzed. (A) Detection of histones and other proteins using PVDF membranes boiled in water for different periods of time. (B, C) Immunoblots of 3 μg lysates of untreated and Trichostatin A (TSA, 18 h)-treated H460 cells using standard and boiled (30 min) PVDF membranes.

Figure 2.

Figure 2

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Immunoblots of IMR90 whole cell lysates using standard and boiled PVDF membranes. Proteins were transferred on PVDF membranes using a standard procedure with PierceG2 Fast Blotter. (A) Higher detection sensitivity for histones H2A and H2B on boiled membranes. (B) Higher detection sensitivity of histone H4 and its posttranslational modifications on boiled membranes. (C) Similar detection of histone H3, its PTMs, and typical loading controls on boiled membranes.

Immunodetection of Recombinant Histones on PVDF Membranes

The observed poor detectability of cellular histones on standard PVDF membranes could have been affected by the presence of other cellular proteins, other core histones (H3, H2A and H2B all cluster around 17 kDa marker), and/or their PTMs. To exclude these factors, we examined human recombinant histones whose purity and loading was verified by Coomassie staining of SDS-PAGE gels (Figure 3A). Using the same semidry transfer conditions and the same antibodies as in above studies with cellular histones, we confirmed a much higher sensitivity of boiled PVDF membranes for detection of pure histones H2AX (with three Abs), H2AZ, H2A.1, H2A (two Abs), H2B (two Abs), and H4 (two Abs). (Figure 3B). Similar to cellular samples, the signals for pure histone H3.1 (main histone H3 form) on standard and boiled PVDF membranes were similar. Antibodies for H3.3 recognized both this variant and canonical histone H3.1 with the same sensitivity irrespective of membrane boiling (Figure 3C). This cross-reactivity indicates that the signal with the anti-H3.3 antibody in cellular samples reflected binding to both H3 and H3.3 (Figure 1C). Thus, poor recognition of several histones by antibodies on standard PVDF membranes largely reflects intrinsic properties of these proteins.

Figure 3.

Figure 3

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Immunoblots of recombinant human histones using standard and boiled PVDF membranes. Duplicate sets of histones (50 ng/lane) were separated on 12% SDS-PAGE and transferred on PVDF membranes using PierceG2 Fast Blotter. (A) Coomassie-stained gel of purified histones after SDS-PAGE. (B, C) Immunoblots of purified human histones using standard and boiled PVDF membranes.

PVDF Immunoblotting of Cellular Histones under Different Protein Transfer Conditions

The observed above differences in sensitivity of many antihistone antibodies on standard vs boiled PVDF membranes were detected after a semidry transfer using a proprietary commercial buffer supplemented with 12% ethanol. Next, we tested whether buffer composition and electric settings have an impact on PVDF boiling effects. We found that omission of ethanol from the standard transfer procedure retained strong beneficial effects of PVDF boiling on detection of the majority of tested histones (H4, H2AX, H2AZ, H2Aub1) although this protocol decreased the overall sensitivity of detection for histones and loading controls (as exemplified by weak signals for H2A.1 and actin) even after long exposure times (Figure S1A). As in the standard protocol, ethanol-free transfer did not result in noticeable effects of PVDF boiling on the detection of histone H3 and loading controls. However, the absence of ethanol in the transfer buffer eliminated the beneficial effect of boiling for histone H2B. Another semidry transfer without ethanol using a lower current and a longer time (1 h) produced results that were very similar to those with the short 13 min transfer without ethanol (Figure S1B). Specifically, H4, H2A1, H2AX, H2AZ and H2Aub1 signals were much higher on boiled PVDF membranes, whereas histone H2B was detected with a similar sensitivity. In another test of semidry conditions, we replaced the proprietary buffer with Towbin buffer+12% ethanol and ran a slow 1 h transfer (Figure S1C). We found that this semidry protocol also required boiling of PVDF membranes for the high-sensitivity detection of three tested histones: H4, H2A.1, and H2Aub1. Thus, with the exception of variable results for H2B, the detection of all other histones showed much greater sensitivity on boiled PVDF membranes under four different semidry protein transfer conditions.

Another popular approach for the protein transfer from SDS-PAGE gels onto membranes is overnight wet electroblotting, which mostly uses Towbin buffer with alcohol (methanol or ethanol). We found that boiling of PVDF membranes after wet transfers with Towbin buffer containing or lacking ethanol also strongly enhanced detection of histones H4 and H2A.1 and more moderately, detection of H2Aub1 (Figure S2A,B). Although the magnitude of the boiling effect for H4 and H2A.1 remained approximately the same for + ethanol and -ethanol wet transfers, the overall sensitivity of detection of H4 and H2A.1, but not H2Aub1 and two loading controls, was much better with the ethanol-containing transfer buffer, as evidenced by the exposure times of the membranes. The third wet transfer procedure that we tested was Bjerrum Shaefer-Nielsen buffer without alcohol. The results again showed dramatically higher sensitivities of boiled PVDF membranes for histones H4 and H2A.1 and more moderate improvements for H2Aub1 (Figure S2C). Overall, boiling of PVDF membranes strongly enhanced detection sensitivities for histones H4 and H2A under a total of seven transfer conditions (four semidry and three wet procedures) and, depending on the transfer protocol, produced strong, moderate, or no effects on detection of histones H2B and H2Aub1. Although exclusion of alcohol from semidry transfer buffers eliminated the differences for H2B detection between standard and boiled membranes, alcohol-free transfers were clearly suboptimal (low overall sensitivity due to a poor transfer efficiency and frequent distortions of bands and gels) for the majority of other histones and some loading controls.

Immunoblotting of Histones on PVDF vs NC Membranes

A previous study examining technical factors affecting isolation and detection of histones did not find marked differences between NC and PVDF membranes using immunoblotting for acetylated histone H3 (H3K56ac).27 In light of the observed above different responses of histone H3 (insensitive) and other core histones (sensitive) to PVDF membrane boiling, we next examined the relative sensitivity of PVDF and NC membranes for immunodetection of all major histones using a popular semidry procedure with the Bio-Rad Trans-Blot Turbo apparatus. In agreement with the reported findings for H3K56ac,27 immunoblotting for N-terminus acetylated histone H3 (H3K9ac and H3K27ac) found no major differences between PVDF and NC membranes with a modest positive effect of boiling on NC membranes (Figure 4A). Detection of histone H3 with antibodies raised against its C-terminal sequence showed no boiling effect on PVDF and NC membranes, but the signal was substantially stronger on both NC membranes. The linker histone H1 and its H1.2 variant were detected with a higher sensitivity on boiled PVDF vs standard PVDF membranes and on NC membranes vs PVDF membranes (especially relative to standard PVDF). Consistent with the above results using other transfer conditions, boiling of PVDF membranes strongly enhanced the detection sensitivity of histones H2A, H2B, H4 and their acetylated forms (Figure 4B). In general (the sole exception was H2BK5ac), standard NC membranes were even more sensitive for the detection of these histones than boiled PVDF membranes. Boiling of NC membranes further increased the detection sensitivity of a majority of the tested histones. Boiling of PVDF membranes and the use of either standard or boiled NC also eliminated the under-detection of total H2B from the hyperacetylated chromatin [as also seen on PVDF membranes after different transfer conditions (Figure 1B)]. Unlike histones, the majority of loading controls showed a similar sensitivity on both types of membranes, whereas PCNA had a stronger signal on the standard PVDF membrane (Figure 4C). With the exception of increased PCNA detection on NC membranes, boiling generally moderately decreased signals on PVDF membranes and had no effects on NC membranes. The size of the tested loading controls was in the range of 32–50 kDa, which is >2-times larger than that of core histones. To investigate whether the small size of proteins makes them poorly detectable on standard PVDF membranes and requires boiling for increased sensitivity, we compared immunoblotting results on PVDF and NC membranes for two small proteins: ubiquitin (8.5 kDa predicted molecular weight, moves as ∼11 kDa band) and thioredoxin 1 (TRX1, 11.7 kDa). We found that both proteins had higher signals on standard PVDF than on standard NC membranes, with especially dramatic differences for ubiquitin (Figure 4C). Boiling strongly increased the detection sensitivity of TRX1 on both membranes whereas a positive effect of boiling for ubiquitin was only observed on NC membranes. The opposite trends in the detection sensitivity for ubiquitin (high on PVDF) and histones H2A/H2B (high on NC) help explain moderate improvements in detection of monoubiquitinated vs unmodified H2A and H2B on NC membranes (Figure 4B). Taken together, insensitivity of NC membranes for small nonhistone proteins, lack of boiling and membrane type effects for acetylated histone H3 and enhanced detection of a larger histone macroH2A1.2 (40 kDa) due to boiling and on NC membrane indicate that the small size of H2A, H2B and H4 alone was not responsible for their poor detection on PVDF membranes and in the absence of boiling.

Figure 4.

Figure 4

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Comparison of standard and boiled PVDF and NC membranes for detection of cellular histones and selected other proteins. Duplicate sets of samples were transferred on PVDF or NC membranes using Bio-Rad Trans-Blot Turbo and membranes were cut in two pieces. One piece of each membrane underwent washes with TBST while other halves were boiled in water for 30 min prior to TBST washes. Whole cell lysates of untreated and Trichostatin A (TSA, 18 h)-treated H460 cells were loaded at 3 μg/lane. (A) Immunoblots of histones H3 and H1. (B) Immunoblots of histones H2A, H2B and H4. (C) Immunoblots of nonhistone proteins.

Detection of γ-H2AX on Standard and Boiled PVDF Membranes

Ser139-phosphorylated histone H2AX (known as γ-H2AX) is the most frequently used biochemical marker of genotoxic stress in cells.21,22 A rapid formation of γ-H2AX was initially discovered in response to DNA ds-breaks produced by ionizing radiation.31 DNA ds-breaks originating from both direct and indirect mechanisms are known to lead to the extensive H2AX phosphorylation.21,22,32−34 The production of γ-H2AX has also been detected in cells experiencing genotoxic stress even in the absence of detectable DNA ds-breaks.35,36 In light of its high popularity as a marker of DNA ds-breaks and genotoxic stress, we next explored immunodetection of γ-H2AX on standard and boiled PVDF membranes using different protein transfer conditions and antibodies. The phospho-Ser139 epitope in γ-H2AX is located near the very end of the C-terminus (three amino acids away), which is expected to make it readily accessible to antibodies. We tested H460 cells treated with the radiomimetic bleomycin, the topoisomerase II poison etoposide and the topoisomerase I inhibitor camptothecin as inducers of γ-H2AX. Bleomycin and etoposide produce DNA ds-breaks in all cell cycle phases whereas camptothecin-stimulated ds-breaks are less frequent as they arise only in S-phase through collapse of replications forks at the sites of ss-DNA breaks containing trapped TOP1.37 Similar to our above findings with the immunodetection of histone H2AX, boiling of PVDF membranes resulted in a dramatically higher detection sensitivity of γ-H2AX by all three tested antibodies under different protein transfer conditions (Figure 5A-C). Quantitative analysis of bands for bleomycin-treated samples typically found >20-fold increases in the sensitivity of γ-H2AX detection on boiled membranes which showed near saturation-level intensities under exposure conditions with barely or completely undetectable γ-H2AX on the standard membranes. Polyclonal antibodies showed somewhat smaller differences between boiled and standard membranes under one of two semidry transfer conditions (Figure 5A, bottom image), which probably reflects a better accessibility of the γ-H2AX epitope for some clones.

Figure 5.

Figure 5

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Immunoblots of γ-H2AX on standard and boiled PVDF membranes. Duplicate sets of insoluble proteins (1 μg) from H460 cells treated for 1 h with 2 μM bleomycin (Bleo), 80 μM etoposide (Eto) or 1 μM camptothecin (CPT) were separated on 12% gels and then transferred on PVDF membranes, which were boiled (30 min) or not boiled and probed with three different antibodies recognizing γ-H2AX. (A) Detection of γ-H2AX with polyclonal antibodies from Cell Signaling (2577) on standard and boiled PVDF membranes after different transfer procedures. (B) Immunoblots for γ-H2AX with monoclonal antibodies from Millipore (05–636) on standard and boiled PVDF membranes. (C) Immunoblots for γ-H2AX with monoclonal antibodies from Abcam (ab26350) on standard and boiled PVDF membranes.

Comparison of γ-H2AX Detection on Standard and Boiled PVDF and NC Membranes

In light of strong positive effects of both boiling and NC membrane use on the detection of histone H2AX with antibodies recognizing its C-terminal regions (Figure 4B), we next examined how these factors affect sensitivity of immunoblotting for γ-H2AX with two popular monoclonal antibodies after two common protein transfer procedures. We found that boiling of membrane-bound proteins strongly enhanced γ-H2AX detection under all tested conditions and ranged from several-fold increases for NC membranes to >100-fold on PVDF membrane (Figure 6). The use of standard NC membranes produced stronger bands than on boiled PVDF membranes at three out of four tested conditions. Overall, the combination of boiling and NC membranes gave >100-times higher detection sensitivity relative to standard PVDF membranes at all conditions. For one antibody (Abcam, ab26350), the improvement in the detection sensitivity was estimated to be >1000-fold at both semidry and wet transfer conditions (Figure 6A,C).

Figure 6.

Figure 6

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Comparison of γ-H2AX detection on standard and boiled PVDF and NC membranes. Duplicates sets of insoluble proteins (1 μg) from control and 2 μM bleomycin (Bleo, 1 h)-treated H460 cells were separated on 12% gels and then transferred on PVDF or NC membranes, which were then boiled (30 min) or not boiled and probed with two indicated monoclonal antibodies recognizing γ-H2AX. (A,B) Detection of γ-H2AX on standard and boiled PVDF or NC membranes after semidry transfer using the Bio-Rad Trans-Blot Turbo Transfer apparatus. (C,D) Immunoblots of γ-H2AX on PVDF and NC membranes after wet transfer using Towbin buffer containing 20% methanol. *-signal on the standard PVDF membrane was undetectable and the fold change was calculated from the estimated minimal detection value.

Conclusions

Our examination of different immunoblotting conditions for detection of human histones and their PTMs revealed a very poor epitope availability on PVDF membranes, especially for antibodies targeting histones H2A, H2B, H4 and H1. This problem can be ameliorated by denaturation of PVDF membrane-bound proteins by a simple boiling procedure, which increased the sensitivity of detection for many histones by >20-times and for some antibodies, also enhanced specificity for the main target (canonical H2A with pan-variant specific H2A antibodies or histone H2B in hyperacetylated samples, for example). In comparison to standard PVDF membranes, standard NC membranes showed a far more superior detection sensitivity for the majority of histones, which was comparable or higher than the detectability on boiled PVDF membranes. Boiling of NC membranes further increased their detection sensitivity for the same histones as on boiled PVDF membranes. Immunoblotting of the genotoxic marker γ-H2AX on boiled NC and after some transfer conditions, also on boiled PVDF membranes gave >100-times higher sensitivity relative to standard PVDF membranes. Strongly beneficial effects of boiling and NC on the epitope availability were observed for antihistone antibodies targeting both N-terminal (location of the majority of small PTMs) and C-terminal regions (total histones and γ-H2AX).

Stripping of protein-bound SDS during transfer from gels facilitates protein binding to membranes but it also allows a partial renaturation of proteins.28,29 Partially denatured proteins can exist in a relatively compact state38 and additional denaturation (such as via boiling) should create a more highly unfolded state that permits a greater accessibility of epitopes to antibodies. Some proteins are also known to bind to membranes in layers, restricting accessibility of antibodies to the protein regions at the sites of membrane binding and through steric hindrance in multilayered arrangements of proteins on the membranes.25 Thus, it is possible that boiling acts as a disruptor of weak histone-membrane contacts and dense layered structures, exposing histone epitopes and diminishing the steric hindrance for antibody binding. A higher protein-binding capacity of PVDF membranes, which is often mentioned as its advantage, likely facilitates weak multipoint interactions with histones, thereby promoting the formation of layered protein structures, which can explain greater beneficial effects of boiling on histone detection on PVDF membranes.

Glossary

Abbreviations

PTM

posttranslational modification

TBST

Tris-Buffered Saline with Tween

TSA

Trichostatin A

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.4c00307.

  • Additional Western blots demonstrating PVDF membrane boiling effects on detection of histones after semidry and wet transfer procedures (PDF)

Author Contributions

CRediT: Casey Krawic investigation, methodology, writing - review & editing; Michal W. Luczak investigation, writing - review & editing; Anatoly Zhitkovich conceptualization, formal analysis, funding acquisition, project administration, writing - original draft.

This work was supported by grants ES031979, ES031002, and ES028072 from the National Institute of Environmental Health Sciences.

The authors declare no competing financial interest.

Supplementary Material

tx4c00307_si_001.pdf (1.7MB, pdf)

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Supplementary Materials

tx4c00307_si_001.pdf (1.7MB, pdf)

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